⎇ⓥ⺖㗴ฬ㧦↢૕㜞ಽሶ࠲ࡦࡄࠢ⾰ߩ X ✢⚿᥏᭴ㅧ⸃ᨆ
⎇ⓥઍ⴫⠪㧦ባୖ ᢥᄦ㧔ᣣᧄᄢቇකቇㇱ⥄ὼ⑼ቇ♽㧕⎇ⓥᓥ੐⠪㧦ባୖ ᢥᄦ㧔ᣣᧄᄢቇකቇㇱ⥄ὼ⑼ቇ♽㧕 ᪀↰ 㓉↢㧔ᣣᧄᄢቇℂᎿቇㇱ PDF㧕౒ห⎇ⓥ⠪㧦㜞ᧁ ዏ㧔᧲ർᄢቇᄢቇ㒮↢๮⑼ቇ⎇ⓥ⑼㧕
 ᅏ ᔘᱞ㧔ᣣᧄᄢቇ↢‛⾗Ḯቇㇱㄘ⧓ൻቇ⑼㧕  ᧖↰ඳᤘ㧔╳ᵄᄢቇ↢‛⑼ቇ♽㧕
㋈ᧁ⍮ᒾ㧔㜞⍮ᄢቇℂቇㇱ↢ൻቇᢎቶ㧕ᓟ⮮ኼᄦ㧔ᓼፉᄢቇ✚ว⑼ቇㇱ↢๮⑼ቇᢎቶ㧕⍹Ꮉ⚔৻㧔ᣣᧄᄢቇකቇㇱ⮎ℂቇᢎቶ㧕‫⎇ޣ‬ⓥ⋡⊛‫ޤ‬ ᣣᧄᄢቇ㊂ሶ⑼ቇ⎇ⓥᚲߪᐔᚑ 12 ᐕᐲޯᐔᚑ 16 ᐕᐲߩᢥㇱ⑼ቇ⋭ߩቇⴚࡈࡠࡦ࠹ࠖࠕߦㆬቯߐ
ࠇߚ‫ޕ‬ห⎇ⓥᚲ㔚ሶ✢೑↪⎇ⓥᣉ⸳ߦ↢૕㜞ಽሶ X ✢⚿᥏᭴ㅧ⸃ᨆታ㛎ቶࠍ┙ߜ਄ߍ㧘ቇౝਗ߮ቇ
ᄖߩ౒ห೑↪⎇ⓥ⠪ߣ⚿᥏૞⵾߆ࠄ᭴ㅧ⸃ᨆ߹ߢߩᛛⴚࠍ౒᦭ൻߔࠆ‫ߦ߼ߚߩߘޕ‬ᰴߩ 3 ߟߩࡊࡠ࠮ࠬࠍน෸⊛ߔߺ߿߆ߦ㆐ᚑߔࠆߎߣࠍ╙ 1 ⋡ᮡߣߔࠆ‫ޕ‬1 ᮡḰ࠲ࡦࡄࠢ⾰ߣߒߡ࡝࠱࠴࡯ࡓࠍᜰᮡߦ㧘ዉ౉ߒߚᯏེ㘃ࠍᬌ⸽ߔࠆ‫ޕ‬หᤨߦ㧘X ✢⚿᥏⸃ᨆᛛⴚࠍ⠌ᓧߔࠆ‫ޕ‬
2 ࠱࠙ࠟࡔߩࡋࡕࠣࡠࡆࡦ㧔Hb㧕ࠍᣂⷙ࠲ࡦࡄࠢ⾰ߦ㧘⚿᥏ൻ߆ࠄ᭴ㅧߩ♖ኒൻਗ߮᭴ㅧࡕ࠺࡞
ࠍ᳿ቯߔࠆ‫ޕ‬3 ⺰ᢥࠍ౏⴫ߔࠆ‫ޕ‬ 1㨪3 ࠍ㆐ᚑߒߚ਄ߢ㧘ߐࠄߦ㧘X ✢⚿᥏᭴ㅧ⸃ᨆ⎇ⓥ⠪ߦኻߔࠆ⎇ⓥᡰេ૕೙ࠍ゠㆏ߦߩߖ㧘౒ห⎇ⓥਗ߮೑↪ታ㛎ᣉ⸳ߣߒߡ౒หᵴേࠍዷ㐿ߔࠆ‫ޕ‬ ߹ߚ㧘ห⎇ⓥᚲ㔚ሶ✢೑↪⎇ⓥᣉ⸳ߢቇⴚࡈࡠࡦ࠹ࠖࠕផㅴ੐ᬺߣߒߡ㐿⊒ߔࠆ㜞ノᐲනᵄ㐳శḮߩࡄ࡜ࡔ࠻࡝࠶ࠢ X ✢శḮߩ㜞ᐲ೑↪⎇ⓥߦㅪଥߒߡ㧘ࡄ࡜ࡔ࠻࡝࠶ࠢ X ✢ࠍ೑↪ߒߚ X ✢⚿᥏᭴ㅧ⸃ᨆߩᛛⴚࠍ㐿⊒ߒ㧘ߘߩᛛⴚ⒖ォࠍߪ߆ࠆ‫ޕ‬‫⎇ޣ‬ⓥ᭎ⷐ‫ޤ‬
ᐔᚑ 12 ᐕᐲ߆ࠄᐔᚑ 16 ᐕᐲߩ⎇ⓥ᭎ⷐߪᰴߩㅢࠅߢ޽ࠆ‫ޕ‬1 ⎇ⓥḰ஻ᐔᚑ 12 ᐕ 4 ᦬㨪ᐔᚑ 13 ᐕ 3 ᦬ ᑪደߩᑪ⸳‫ޕ‬ᐔᚑ 13 ᐕ 3 ᦬┫Ꮏᑼ
ᐔᚑ 13 ᐕ 6 ᦬ X ✢⚿᥏᭴ㅧ⸃ᨆታ㛎ቶߩ⸳஻ቢੌ‫ޕ‬⎇ⓥᡰេ⠪㧔᪀↰㓉↢ඳ჻㧕㓹↪‫ޕ‬
2 ࡝࠱࠴࡯ࡓߩ⚿᥏ൻਗ߮᭴ㅧ⸃ᨆ㧔ዉ౉ߒߚᯏེ㘃ߩᬌ⸽ߣ⚿᥏᭴ㅧ⸃ᨆᛛⴚߩ⠌ᓧ㧕
ᐔᚑ 13 ᐕ 6 ᦬㨪ᐔᚑ 14 ᐕ 5 ᦬㧔⚳ੌ㧕
3 ᣂⷙ࠲ࡦࡄࠢ⾰࠱࠙ࠟࡔߩ Hb D ߩ⚿᥏᭴ㅧ⸃ᨆ ᐔᚑ 14 ᐕ 5 ᦬㨪ᐔᚑ 15 ᐕ 8 ᦬㧔⚳ੌ㧕
4 ቇᄖ౒ห⎇ⓥࠕࠞࡓࠪߩ Hb D ߩ⚿᥏᭴ㅧ⸃ᨆ ᧲ർᄢቇᄢቇ㒮 㜞ᧁዏᢎ᝼ߣߩ౒ห⎇ⓥ ᐔᚑ 15 ᐕ 11 ᦬ޯᐔᚑ 16 ᐕ 3 ᦬㧔⚳ੌ㧕
5 ቇᄖ౒ห⎇ⓥߩ⛮⛯ 㜞⍮ᄢቇℂቇㇱ ㋈ᧁ⍮ᒾᢎ᝼ߣߩ౒ห⎇ⓥ㧔ࠪࡠ࠙࡝ࠟࠗߩ Hb ߩ᭴ㅧ⎇ⓥ㧕 㕒ጟᄢቇℂቇㇱ ┻ౝᶈᤘഥᢎ᝼ߣߩ౒ห⎇ⓥ㧔ࠕࡎࡠ࡯࠻࡞ߩ Hb ߩ⎇ⓥ㧕 ╳ᵄᄢቇ↢‛⑼ቇ♽ ᧖↰ඳᤘᢎ᝼ߣߩ౒ห⎇ⓥ㧔ࠞࡉ࠻ࠟ࠾ߩࡋࡕࠣࡠࡆࡦߩ᭴ㅧ⎇ⓥ㧕㧢 ᣉ⸳೑↪⎇ⓥߩ㐿ᆎ 㤗ᏓᄢቇⅣႺ଻ஜቇㇱ ૒ୀື㇢ᢎ᝼ߩ X ✢᭴ㅧ⸃ᨆታ㛎 ╳ᵄᄢቇ↢‛⑼ቇ♽ ᴧ↰ᴦᢎ᝼ߩ X ✢᭴ㅧ⸃ᨆታ㛎‫ޤ߼ߣ߹ޣ‬ᢥㇱ⑼ቇ⋭ቇⴚࡈࡠࡦ࠹ࠖࠕផㅴ੐ᬺߩ⎇ⓥᐕᐲ㧔ᐔᚑ 12 ᐕᐲޯᐔᚑ 16 ᐕᐲ㧕ߦ߅ߌࠆ⎇ⓥᚑ
ᨐߪᰴߩㅢࠅߢ޽ࠆ‫ޕ‬1 ᣣᧄᄢቇ㊂ሶ⑼ቇ⎇ⓥᚲ㔚ሶ✢೑↪⎇ⓥᣉ⸳ߦ↢૕㜞ಽሶ X ✢⚿᥏᭴ㅧ⸃ᨆታ㛎ቶࠍ᭴▽ߒߚ‫ޕ‬
2 X ✢⚿᥏᭴ㅧ⸃ᨆታ㛎ࠍታᣉߢ߈ࠆࠃ߁ߦห਄ታ㛎ቶࠍᢛ஻ߒߚ‫⚿ߩߘޕ‬ᨐ㧘⚿᥏૞⵾߆ࠄ᭴
ㅧߩ♖ኒൻ߹ߢߩ૞ᬺࠍታᣉߢ߈ࠆ‫ޕ‬
3 X ✢⚿᥏᭴ㅧ⸃ᨆታ㛎ࠍ⚳ੌߒ㧘┙૕᭴ㅧߩࡕ࠺࡞ࠍ᳿ቯߒߚ࠲ࡦࡄࠢ⾰ߦߪ㧘࠱࠙ࠟࡔߩ Hb
D㧘ࠕࠞࡓࠪߩ Hb V ߣ Hb VII㧔ᛩⓂḰ஻ਛ㧕߇޽ࠆ‫ߚ߹ޕ‬㧘ࠪࡠ࠙࡝ࠟࠗߩ 2 ⒳㘃ߩ Hb㧘ࠕࡎࡠ࡯࠻࡞ߩ 2 ⒳㘃ߩ Hb㧘ࠞࡉ࠻ࠟ࠾ߩࡋࡕࠪࠕ࠾ࡦߥߤߩ⚿᥏ൻࠍㅴ߼ߡ޿ࠆ‫ޕ‬
4౒ห⎇ⓥ෸߮ᣉ⸳೑↪⎇ⓥ߇㐿ᆎߐࠇߡ㧘ᚑᨐࠍ޽ߍߡ޿ࠆ‫ޕ‬⎇ⓥ⋡⊛ߣߒߡឝߍߚ⸘↹ߪ㧘ࡄ࡜ࡔ࠻࡝࠶ࠢ X ✢ࠍ೑↪ߒߚ X ✢⚿᥏᭴ㅧ⸃ᨆߩᛛⴚࠍ㐿⊒ߔࠆߎߣ㧔⃻࿷㐿⊒ਛ㧕એᄖߔߴߡ㆐ᚑߒߚ‫⃻ޕ‬࿷㧘౒ห⎇ⓥߣᣉ⸳೑↪⎇ⓥࠍዷ㐿ߒߡ޿ࠆ‫ޕ‬‫⎇ޣ‬ⓥᬺ❣╬‫ޤ‬
࡮⊒⴫⺰ᢥ
(1)

F. Shishikura and K. Takami (2001) The amino acid sequences of the D- and E-globin chains ofhemoglobin from the Aldabra giant tortoises, Geochelone gigantea.

(2)

Zoological Science 18, 515-526.

F. Shishikura (2002) The primary structure of hemoglobin D from the Aldabra giant tortoise,
Geochelone gigantea.

Zoological Science 19, 197-206.

(3)ባୖᢥᄦ (2002) ↢૕㜞ಽሶ┙૕᭴ㅧ⸃ᨆታ㛎ቶߩ┙ߜ਄ߍ. ᣣක㔀⹹ 61, 179.

(4)ባୖᢥᄦ (2002) Ῥ⯻㘃ߩࡋࡕࠣࡠࡆࡦ㧦࠱࠙ࠟࡔ㧔Geochelone gigantea ߣ Geochelone nigra㧕ࠣࡠࡆࡦߩಽሶㅴൻ. ᣣක㔀⹹ 61, 263 – 276.

(5)

T. Kuwada, T. Hasegawa, I. Satoh, K. Ishikawa and F. Shishikura (2003) Crystallization andpreliminary x-ray diffraction study of hemoglobin D from the Aldabra giant tortoise, Geochelone
gigantea. Protein and Peptide Letters 10, 422-425.

(6)

F. Shishikura (2004) Leech hemoglobin: primary structures of four kinds of globins from Haemadipsa
zeylanica var. japonica. Micron 35, 123-125.

(7)

F. Shishikura (2004). The complete amino acid sequences of four globins from the land leech
Haemadipsa zeylanica var. japonica. Comparative Biochemistry and Physiology 137B, 443-454.

࡮࿑ᦠ
(1)

ባୖᢥᄦ㧘᪀ᴛᷡ᣿㧔2002㧕ⴊᶧ࡮ࡋࡕ࡝ࡦࡄᶧ㧛ή⣄ᬁേ‛ ↢‛ቇ࠺г࠲ᄢ⊖⑼੐ౖ㧔⍹ේൎᢅ ✬㓸ઍ⴫㧕ᦺୖᦠᐫ㧘pp. 1019 – 1093, ᧲੩

࡮ቇળ⊒⴫
(1)

᪀↰㓉↢㧘ባୖᢥᄦ (2002) ࠕ࡞࠳ࡉ࡜࠱࠙ࠟࡔ㧔Geochelone gigantea㧕ߩࡋࡕࠣࡠࡆࡦ D ߩ⚿᥏ൻ߅ࠃ߮ X ✢࿁᛬. ᣣᧄ⚿᥏ቇળᐔᚑ 14 ᐕᐲᐕળ⻠Ṷⷐᣦ㓸㧘21 㗁.

(2)᪀↰㓉↢㧘ባୖᢥᄦ (2003) ࠕ࡞࠳ࡉ࡜࠱࠙ࠟࡔ㧔Geochelone gigantea㧕ߩࡋࡕࠣࡠࡆࡦ D ߩ
X ✢⚿᥏⸃ᨆ. ᣣᧄ⚿᥏ቇળᐔᚑ 15 ᐕᐲᐕળ⻠Ṷⷐᣦ㓸㧘122 㗁.

(3)

F. Shishikura (2003) Leech hemoglobin: Primary structures of four kinds of globins from Haemadipsa
zeylanica var. japonica. XIIIth International Conference on Invertebrate Dioxygen Binding Proteins.
Johannes Gutenberg Universität Mainz, Mainz, Germany, September.

(4)

T. Kuwada, T. Hasegaw S. Sato, I. Sato, K. Ishikawa, T. Takagi and F. Shishikura (2004)
Tokunagayusurika akamusi (Diptera) hemoglobin: Crystallographic analyses of Hb V and Hb VII at 1.65
Å and 1.50 Å resolution, respectively. Zoological Science 21, 1327.

(5)

F. Shishikura and H-a, Takeuchi (2004) Ambystoma mexicanum (axolotl, Amphibia) hemoglobin: The
complete amino acid sequences of two types of D globins and one E globin. Zoological Science 21, 1327.

࡮⊒⴫⺰ᢥ(ᅏ ᔘᱞ࡮⊒⴫ಽ)(1) Suruga, K., Murakami, K., Taniyama, Y., Hama, T., Chida, H., Satoh, T., Yamada, S., Hakamata, W.,
Kawachi, R., Nishio, T. and Oku, T., A novel microperoxidase activity: methyl-viologen-linked nitrite
reducing activity of microperoxidase, Biochemical and Biophysical Research Communications, 315,815-822 (2004)(2) Ishida, M., Dohmae, N., Shiro, Y., Oku, T., Iizuka, T. and Isogai, Y., Design and synthesis of de novo
cytochrome c, Biochemistry, 43(30), 9823-9833 (2004)(3) Suruga, K., Nagasawa, N., Yamada, S., Satoh, T., Kawachi, R., Nishio, T., Kume, T. and Oku, T.,
Radiation-induced enhancement of nitrite reducing activity of cytochrome c, Journal of Agriculturaland Food Chemistry, 51(23), 6835-6843 (2003)(4) Nishio, T., Hoshino, S., Kondo, A., Ogawa, M., Matsuishi, Y., Kitagawa, M., Kawachi, R. and Oku, T.,
Glycosidase-catalyzed deoxy oligosaccharide synthesis. Practical synthesis monodeoxy analogs of ethyl
E-thioisomaltoside using Aspergillus niger D-glucosidase, Bioscience Biotechnology Biochemistry,67(5), 1024-1029 (2003)

ZOOLOGICAL SCIENCE 18: 515–526 (2001)© 2001 Zoological Society of Japan

The Amino Acid Sequences of the α- and β-Globin Chains of
Hemoglobin from the Aldabra Giant Tortoises,
Geochelone gigantea
Fumio Shishikura1* and Kazutoshi Takami2
1

Department of Biology, Nihon University School of Medicine,Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610
2
Osaka Municipal Tennoji Zoo, 1-108, Chausuyama,
Tennoji, Osaka 543-0063, Japan

ABSTRACT—Tetrameric hemoglobins (Hbs) A and D were isolated from red blood cells of the Aldabra giant
tortoises, Geochelone gigantea, by a hydrophobic interaction chromatography. After reduction andS-pyridylethylation, two sets of two types of α-chains (α-1 and α-2) and one β-chain were purified from the
major Hb A and minor Hb D in molar ratios of about 1:1:2, respectively, by a reversed-phase column chromatography. The complete amino acid sequences of the three globin-chains from Hb A were determined: 141
amino acid residues for the two α-chains and 146 amino acid residues for the β-chain. Using computer
analysis (amino acid maximum homology), the two α-chains shared a 96.5% sequence identity and had low
sequence identities (37.8% for α-1 and 35.8% for α-2) with the β-chain of the same species, G. gigantea.
We constructed a phylogenetic tree of 28 primary globin structures from Reptilia (7 species of squamates,
4 species of turtles, 3 species of crocodiles and 1 species of sphenodontids), including the three globins of G.
gigantea Hb A. The following results were obtained: (1) The two terrestrial species of Geochelone (G. gigantea
and G. carbonaria) were closely related: 139 amino acid residues (95.2%) of the two β-globin chains wereconserved; (2) Based on the divergence patterns of globin-chains, the sea turtle Caretta caretta was shown
to be unusual relatedness form the groups of terrestrial and freshwater species in turtles. The molecular
relationships appearing on the phylogenetic tree also support the traditional classification of reptiles andpartly confirm previous molecular studies of reptilian hemoglobin evolution.

INTRODUCTION
Hemoglobin, the major respiratory protein, has been
extensively investigated in animals, plants, protozoans, fungi
and bacteria (Keilin, 1956; Kleinschmidt and Sgouros, 1987).
At the molecular level the protein has provided much information in both functional and evolutionary aspects (Bunn andForget, 1986; Goodman et al., 1988; Vinogradov et al., 1993).
In reptiles, to our knowledge, 35 globin-chains from 19 species have been sequenced (Gorr et al., 1998; Kleinschmidt
and Sgouros, 1987; Fushitani et al., 1996). Among these studies, there are four investigations on turtle hemoglobin: onefor a land tortoise, Geochelone carbonaria (Bordin et al., 1997),
one for a sea turtle, Caretta caretta (Petruzzelli et al., 1996),
and two for freshwater turtles, Chrysemys picta bellii(Rücknagel et al., 1984) and Phrynops hilarii (Rücknagel etal., 1984).
This study aimed to establish the complete primary structures of both types of globins (α and β) from the land tortoises* Corresponding author: Tel. +81-3-3972-8111;
FAX. +81-3-3972-0027.
E-mail: fshishi@med.nihon-u.ac.jp.

Geochelone gigantea, and analyze phylogenetic relationships
among reptiles including two species of Geochelone, G.
gigantea and G. carbonaria. First, the two hemoglobin components (Hb A and Hb D) were separated from G. gigantea
hemoglobin under native conditions and purified constitutive
polypeptide globin-chains from each of the two hemoglobin
components for use in analyzing their primary structures. This
study provides complete primary structures of the three globinsfrom Hb A of G. gigantea. Here, we also describe a phylogenetic tree constructed for 14 complete amino acid sequencesof both α-type globins and β-type globins of reptilian hemoglobins, including the two α-globins and one β-globin of G.
gigantea Hb A. The phylogenetic tree supports previous studies on the classification, phylogeny and molecular evolutionof reptiles (Benton, 1990, Fushitani et al., 1996: Gorr et al.,1998).
MATERIALS AND METHODS
Materials
Blood from a male Aldabra giant tortoise, G. gigantea, weighing
approximately 36 kg, was collected in heparin-Tris-HCl buffer, pH 8.0,
at Osaka Municipal Tennoji Zoo where the animal died just before

516

F. Shishikura and K. Takami

bleeding. The animal came from the Aldabra Atoll located only about
350 km from the northern coast of Madagascar and about 600 km
away from East Africa’s coast. Acetonitrile, ammonium sulfate,ammonium bicarbonate, tri-n-butyl phosphine, 4-vinyl pyridine andV8 protease (from Staphylococcus aureus strain V8) were purchasedfrom Nakalai Tesque, Inc. (Kyoto, Japan). Trifluoroacetic acid (TFA)
was obtained from Sigma Chemical Co. (St. Louis, MO. USA). Lysyl
endopeptidase (Achromobactor protease I) was purchased from Wako
Pure Chemicals Co. (Tokyo, Japan). Separation columns, Alkyl
Superose column HR5/5 and Resource column (3 ml prepackaged
with source 15 RPC gel matrix), were purchased from Pharmacia
Biotech (Upsala, Sweden), and placed in a fast protein liquid chromatography (FPLC) system (Pharmacia Biotech). All other chemicals
and solvents used were of the most purified grade commercially available.
Preparation of hemoglobin solution
Red blood cells were washed three times in 10 vol. physiologicalsaline, lysed with 1 mM Tris-HCl, pH 8.0, and centrifuged at 3000 × g
for 15 minutes to remove cell debris. All procedures were done at
4°C. The cell lysate, hemoglobin solution, was aliquoted and stored
at –80°C until use.
Separation of hemoglobin components
The hemoglobin solution which had been saturated at 40% byadding 60% saturated ammonium sulfate was subjected to an Alkyl
Superose column equilibrated with 60% saturated ammonium sulfate(183 g/ 500 ml) in 50 mM ammonium bicarbonate, pH 8.0. Elution
was carried out with a gradient of 60– 0% saturated ammonium sulfate in the 50 mM ammonium bicarbonate buffer. The flow rate wasmaintained at 0.5 ml/min and fractions of protein peaks were collected. The fractions were monitored at 415 and 280 nm by spectrophotometers (Model 115, Gilson and UV-1, Pharmacia Biotech).
Protein modification
Reduction and S-pyridylethylation of globins were performed by
the method described previously (Friedman et al., 1970). After eachreaction, the modified protein was dialyzed against 0.1M ammonium
bicarbonate and lyophilized. Finally, the remaining reagents werecompletely removed from the sample by reversed-phase column chromatography on Resource from water containing 0.1% TFA to 80%
acetonitrile containing 0.08% TFA. Flow rates were maintained at 0.5
ml/min. The fractions were monitored at 214 and 280 nm by a spectrophotometer (Model 116, Gilson).
Separation and purification of globin-chains
To separate α-type and β-type globin-chains, the S-pyridylethylated hemoglobin was subjected to a Resource column and eluted
with a 0.1% TFA buffered gradient to 60% acetonitrile in 0.08% TFA.
All fractions were monitored at 214 and 280 nm by a spectrophotometer (Model 116, Gilson). For further purification, re-chromatographyon the Resource column was conducted under shallower gradientconditions as described in our previous report (Shishikura et al., 1987).
Enzymatic digestion
Lysyl endopeptidase digestion was performed essentially as
described (Jekel et al., 1983). Briefly, samples (15– 20 nmoles) of the
S-pyridylethylated protein were first dissolved in 8 M urea andincubated at 37°C for 30 min, followed by the addition of 0.5 M
ammonium bicarbonate at a final concentration of 4 M urea in 0.1 M
ammonium bicarbonate. Lysyl endopeptidase digestion of the sample
was performed at an enzyme/substrate ratio of 1:30 (mol/mol) for 4 hr
at 37°C in 0.1 M ammonium bicarbonate solution, pH 8.2 containing
4 M urea. To obtain overlapping peptides, the sample (20 nmoles)
was digested with the V8 protease at a ratio of 1:100 (w/w, enzyme/
substrate) for 48 hr at 37°C in a 0.1M Tris-HCl solution, pH 8.5 containing 1 M urea.

Peptide separation
All peptides derived from their parent molecules were separatedusing a reversed-phase column, Resource, in a 0.1% TFA buffered
gradient to 60% acetonitrile in 0.08% TFA. Flow rates were maintained at 0.5 ml/min. All fractions were monitored at 214 nm and 280
nm by a spectrophotometer (Model 116, Gilson). Re-chromatography of selected peptides, when necessary, was performed as previously described (Shishikura et al., 1987).
Sequence determination
Sequence analysis was performed using a Shimadzu gas phase
protein sequencer, PPSQ-10, equipped with a PTH-10 amino acidanalyzer (Shimadzu Co., Kyoto, Japan). Phenylthiohydantoin (PTH)derivatives from the sequencer were separated and quantified. PTHcysteine was detected as pyridylethylated-PTH-cysteine, the elution
point of which was determined as described in the manufacturer’s
manual.
Computer analysis
A multiple alignment program, Clustal W (Thompson et al., 1994),
was used in the alignment of 28 primary structures of globins from
Reptilia. Pair-wise distances among the 28 globin sequences wereanalyzed using a computer program PROTDIST stored in the PHYLIP
package (v. 3.51c; Felsenstein, 1993) under the Kimura-formula option. Based on the pair-wise distances, Neighbor-Joining/UPGMA in
NEIGHBOR (Felsenstein, 1993) was used to construct the phylogenetic tree of hemoglobins from Reptilia.

RESULTS AND DISCUSSION
Two components of hemoglobins
The red blood cells of the Aldabra giant tortoises, G.
gigantea, contain two main hemoglobin components, major
and minor, which were successfully separated under native
conditions (50 mM ammonium bicarbonate, pH 8.0) by use of
an Alkyl Superose column HR 5/5 (Fig. 1). Two peaks weredetected at 280 nm and 415 nm. They exist at a ratio of about
5:1 based on chromatogram area calculation. This value mayvary from 5:1 to 5:3 depending on sample preparation.
Braunitzer and coworkers have succeeded in separating two
main components, Hb A and Hb D, from the adult Western
Painted Turtle (Chrysemys picta bellii) by use of polyacrylamide gel electrophoresis under alkaline (pH 8.3) and dissociating conditions (Rücknagel and Braunitzer, 1988). Also otherinvestigators successfully separated two components fromsphenodontid hemoglobin on DEAE at pH 8.5, but in this casethey added 0.1% mercaptoethanol and 0.1% dithiothreitol tothe elution buffer (Abbasi et al, 1988) and lysis buffer of red
blood cells (Weber et al., 1989), respectively. Brittain (1988)
determined the existence of three carbomonoxy-form hemoglobin components, T1, T2 and T3, from Sphenodon punctatus
by using DEAE-Sephadex eluted by a gradient of Tris-HCl
buffer (pH 8.5) versus Bistris-HCl buffer (pH 5.5). Bonilla et al(1994) used preparative isoelectric focusing and agarose gelswith ampholines for separation of intact hemoglobin components. They purified two hemoglobins from the South American snake Riotropical Racer, Mastigodryas bifossatus ,however, whose protein bands were closely separated withisoelectric points of 8.02 and 8.07, respectively. Hence, theAlkyl Superose column, a kind of hydrophobic interaction col-

Primary Structures of G. gigantea globins

517

umn, should provide excellent resolution in separation andpurification of intact hemoglobin components of G. gigantea.
Successful separation of intact hemoglobin components
enables the manufacture of crystals from individual hemoglobin components for future study of the relationships between
physiological functions and crystal structures of hemoglobin
components.
Chain separation
After separation of individual components of Geochelone
hemoglobin, the constituents of the major and minor hemo-

Fig. 1. Alkyl Superose HR5/5 column chromatography of the Aldabra
giant tortoises, G. gigantea, intact hemoglobin. The hemoglobin solution was saturated at 40% by adding 60% saturated ammonium sulfate and applied to an Alkyl Superose HR5/5 column equilibrated with
60% saturated ammonium sulfate buffer (buffer A). After washing withbuffer A, the adsorbed proteins were eluted with linearly decreasing
ammonium sulfate concentration from 60% to 0% in 50 mM ammonium bicarbonate, pH 8.0. Elution was monitored at 280 nm (top) and
415 nm (bottom). Flow rate was maintained at 0.5 ml/min. Bars indicate the pooled fractions of major and minor peak, Hb A and Hb D,
respectively.

Fig. 2. Separation of globin-chains from reduced and Spyridylethylated Hb A on Resource column. A linear gradient wasused between 0.1% TFA in water and 0.08% TFA in 60% acetonitrile
at a flow rate of 0.5 ml/min. Elution was monitored at 280 (top) and
214 nm (bottom). Globins from peaks 1, 2, 3 are α-1, α-2 and β globin-chains, respectively. Bars indicate fractions used for sequencing.

518

F. Shishikura and K. Takami

globins could have been further separated under intact conditions by a reversed-phase column using prepackaged
Resource resins but their resolution proved to be inadequate.
However, after reduction and S-pyridylethylation of thehemoglobin, the major hemoglobin yielded three more widely
separated main peaks and several additional minor peaks ona chromatogram obtained by the same reversed-phase column described above. Many investigators have used reversedphase semi-microbore type columns (Abbasi et al., 1988;
Matsuura et al., 1989; Fushitani et al., 1996; Petruzzelli et al.,1996) or CM-columns (Liu, 1975; Leclercq et al., 1982;
Rücknagel and Braunitzer, 1988; Rücknagel et al., 1988; Islam et al., 1990: Naqvi et al., 1994) for separation of globinchains. Our method described here has a similar efficiency
and gives excellent resolution in separation at high flow rates
with low backpressure (versus flow rates). Figure 2 shows atypical separation profile of three main peaks of globin-chainsfrom the major hemoglobin. The advantages to modify theprotein by reduction and S-pyridylethylation were also true forseparation of globin-chains from the minor hemoglobin (datanot shown). As the results, we prepared six individual globinchains in total from the two hemoglobin components of G.
gigantea.
Table 1 shows the results of the first 20 amino-terminal
amino acid sequences of the six globin-chains. The nomenclature of α- and β-globin chains depends on amino acidsequence similarities to those of the known sequences of reptilian hemoglobins (Kleinschmidt and Sgouros, 1987). Consequently, the major hemoglobin is a kind of Hb A and theminor is Hb D. Two α-types of globins in Hb A or Hb D haveidentical sequences each other so far sequenced (Table 1),
while their patterns on reversed-phase column chromatography are shown distinctly different (Fig. 2, data not shown for
those of Hb D). Figure 2 also shows that two kinds of α-typesof globin-chains (α-1 and α-2) and one β-type globin-chain
are separated at molar ratio of about 1:1:2. This indicates thatthe Hb A is tetrameric hemoglobin comprised α-1/α-2/β2. The
presence of two subtypes of α-globins is completely confirmedby their primary structures shown in Fig. 3.
In birds (Dolan et al., 1997) and crocodiles (Kleinschmidt
and Sgouros, 1987) only one type of β-globin chain has been
demonstrated, while lizards and snakes express two types of
β-globin chains (Fushitani et al., 1996; Gorr et al., 1998). It is
still unknown yet whether the β-globin chains of Hb A and Hb
D from G. gigantea are identical or not, but peptide maps
Table 1. Amino-terminal amino acid residues of six globin chainsfrom the Aldabla giant tortoises, Geochelone gigantea
1

10

20

Hb A α-1 V L T A G D K A N V K T V W S K V G S H
α-2 V L T A G D K A N V K T V W S K V G S H
β V H W T S E E K Q Y I T A L Q W A K V N
1

10

20

Hb D α-1 M L T E D D K Q L I Q H V W E K V L E H
α-2 M L T E D D K Q L I Q H V W E K V L E H
β V H W T S E E K Q Y I T A L Q W A K V N

digested with lysyl endopeptidase (data not shown) and thefirst 20 amino-terminal amino acid residues (Table 1) suggestthat they might be the same. It was also indicated that the
blood of adult Western Painted Turtles, Chrysemys picta bellii(Rücknagel and Braunitzer, 1988) were sharing the same βglobin chains when compared with the two complete aminoacid sequences of β-types of globin-chains from Chrysemys
Hb A and Hb D. On the contrary, in frogs there have beenreported to present two subtypes of β-globin chains (Knöchel
et al., 1983; Patient et al., 1983; Oberthür et al, 1983 and1986). On the numbers of subtypes of β-type globin-chains
among amphibians, reptiles, birds and mammals, thus, reinvestigations are needed, in particular, in view of evolution of
Tetrapoda.
Sequencing and alignments
In general, Amniota (reptiles, birds, and mammals) has
two or more hemoglobin components (Ikehara et al., 1997;
Gorr et al, 1998) which are expressed under different physiological conditions. The presence of α-type (αD) globin-chain
in Hb D is, in particular, of interest in the study of the molecular phylogeny of Amniota because αD-globin chain was firststudied in birds such as chickens (Hagopian and Ingram, 1971;
Brown and Ingram, 1974; Kleinshmidt and Sgouros, 1987).
The nomenclature of Hb A and Hb D was adopted in Ingram’slaboratory (Hagopian and Ingram, 1971; Brown and Ingram,
1974) to describe the various domestic fowl hemoglobins: Theembryonic and adult definitive erythrocytes contain the major
adult (A) hemoglobin and the minor definitive (D) hemoglobin.
Hb D was also reported in the tuatara Sphenodon (Abbasi
et al., 1988). As for the presence of Hb D in turtles, it was firstfound in the adult Western Painted Turtle, Chrysemys picta
bellii (Rücknagel et al., 1984) and the Hilaire’s Sideneck Turtle,
Phrynops hilarii (Rücknagel et al., 1984). This study describes
the presence of Hb D in the Aldabra giant tortoises, G.
gigantea, and also demonstrates the presence of two subtypes of α-type globin-chains. To ascertain the presence of
αD globin-chains in the Hb D of G. gigantea, a study on the
primary structures of the αD globin-chains is in progress (theprimary structure of αD –1 globin chain has been submitted to
the JIPID with an accession number PC7116). To date,
all reptiles sequenced (Abbasi et al., 1988; Matsuura et al.,
1989; Islam et al., 1990; Rücknagel et al., 1988; Abbasi andBraunitzer, 1991; Fushitani et al., 1996) except crocodiles(Leclercq et al., 1981) have been clarified to possess twohemoglobin components, Hb A and Hb D.
Reptilian phylogenetic tree
Geochelone is a unique group among turtles since it
includes two big-size tortoises; G. gigantea, the Aldabra giant
tortoises, and G. elephantopus, the Galapagos giant tortoises.
These species may weigh up to 250 kg and measure 150 cm
over the curve of their carapaces (Jackson, 1984). The
Galapagos tortoises are, in general, known as the world’s largest living tortoises. Recently, Bordin et al (1997) have studied

Primary Structures of G. gigantea globins

519

Fig. 3. Strategies and complete sequences of α-1 (top), α-2 (middle) and β (bottom) globin-chains of Hb A from the Aldabra giant tortoises, G.
gigantea. The complete amino acid sequences of the α-1 (top), α-2 (middle) and β (bottom) globin-chains of G. gigantea Hb A are establishedfrom overlapping peptides and fragments. The residues marked with continuous lines are those identified by Edman degradation method.
Vertical lines represent the beginning and the end of sequencing. Dashed lines indicate the residues not determined but which might be includedin the fragment. Small open circles indicate residues incompletely identified using the fragment. Peptide nomenclatures are as follows: lysyl
endopeptidase, K; V8 protease, E.

520

F. Shishikura and K. Takami

one of the Geochelone species, G. carbonaria, whose carapace size measures at most about 40 cm, and reported theprimary structure of the β-globin deduced from its cDNA
analysis. The distribution range of G. carbonaria stretches
throughout mainland South America: Panama, Colombia, Venezuela, Brazil, Paraguay, and Argentina. Thus, the present
habitats of the three Geochelone species are remote and isolated from one another. Many questions arises, such as “Whendid they diversify from their ancestor?”, “Is there any correlation between diversity of morphological characteristics andevolution of protein structure?”, “How do they differ in theirprimary structures?”, and “How have they adapted proteinfunctions to environmental and physical circumstances? “ Onthese points, comparing the primary structures gives fairly
important clues for understanding and elucidating the evolution and improvements of molecular structures of proteins aswell as genes.

Figure 3 summarizes the strategies used to establish thecomplete amino acid sequences of the three globin-chainsfrom Hb A. Appendix 1 provides the data supporting thesequences of Fig. 3. The two α-globin chains are composedof 141 amino acid residues and the β-chain is composed of
146 residues. All overlaps were quantitatively confirmed by
duplicated analyses of amino acid residues, with the exceptions of residues 22 through 30 and 109 through 120 for α-1
globin-chain, 23 through 30, 61, 63 through 68, and 95 through120 for α-2 globin chain, and 72, 73 and 122 for β-globin chain.
The two α-globin chains and the β-globin chain of G.
gigantea are aligned with those previously reported for reptilian hemoglobins (Appendix 2). When the globin sequencesof the two α-chains and the β-chain of G. gigantea are compared with those of known sequences, there are 19 invariant
amino acids among the 28 globins from reptilian hemoglobins. As for the invariant amino acid residues among the 14

Fig. 4. The phylogenetic tree of 28 reptilian globins. Distance matrices estimated by PRTODIST (option: Kimura formula/data not shown) wasused for construction of a rooted tree by NEIGHBOR under the UPGMA method in the package of PHYLIP (version 3.51c: Felsenstain, 1993).
Branch lengths are proportional to protein distances and shown on the individual branches of the tree. The abscissa is a time scale in Myr (million
years) ago based on the separations of the α- and β- globin chains described by Goodman et al., 1975. The references of globin-chains used in
the present analysis are as follows: 1) this study, 2) this study, 3) Rücknagel et al., 1988, 4) Petruzzelli et al., 1996, 5) Abbasi et al., 1988, 6)
Rücknagel et al., 1988, 7) Abbasi et al., 1988, 8) Fushitani et al., 1996, 9) Islam et al., 1990, 10) Naqvi et al., 1994, 11) Duguet et al., 1974, 12)Leclercq et al., 1981, 13) Leclercq et al., 1981, 14) Leclercq et al., 1982, 15) this study, 16) Bordin et al., 1997, 17) Rücknagel et al., 198, 18)
Petruzzelli et al., 1996, 19) Abbasi et al., 1988, 20) Brittain, 1988, 21) Rücknagel et al., 1988, 22) Abbasi et al., 1991, 23) Islam et al., 1990, 24)
Naqvi et al., 1994, 25) Matsuura et al., 1989, 26) Leclercq et al., 1981, 27) Leclercq et al., 1981, 28) Leclercq et al., 1982.

Primary Structures of G. gigantea globins

α-globins and 14 β-globins from reptiles, there are 44 invariant (31.2%) and 41 invariant (28.1%), respectively. When
compared sequence similarities of globin chains within a species (G. gigantea), the sequence identities of 37.8% (α-1 versus β) and 35.8% (α-2 versus β) were obtained. These
resemble the value (42.5% identity) obtained from the comparison with human α- and β-globin chains (Bunn and Forget,1986). On the contrary, comparing the sequence of the βglobin chain of G. gigantea with that of G. carbonaria, there
are 139 identical amino acid residues. This similarity (95.2%)
coincides with the sequence similarity (96.5%) of the two αglobin chains of G. gigantea. This finding suggests that thetwo species are definitely very closed related to each other,and their protein structures, though they are limited, havesomehow been conserved even when their morphologicalcharacteristics have greatly changed.
Reptilian phylogeny and diversity based on α- and βglobin chains
Phylogenetic analyses of 28 globin-chains including 4
species of Testudinata, 7 species of Squamata (snakes
and lizards), 3 species of Crocodylia and 1 species ofRhynchocephalia were conducted by PROTDIST (Felsenstein,1993). The rooted tree (Fig. 4) deduced by NEIGHBOR under
the UPGMA method (PHYLIPS; Felsenstein, 1993) is highly
correlated at the level of orders with the reptilian traditional
phylogeny established mainly depending on morphologicalcharacteristics (Carroll, 1969; Benton, 1990). This partly supports the previous molecular studies on the evolution of reptilian hemoglobins (Goodman et al., 1975; Fushitani et al., 1996;
Gorr et al., 1998; Vinogradov et al., 1993). The molecular
relationships appearing on our phylogenetic tree are summarized as follows: (1) the two species of Geochelone have separated very recently (estimated to be about 17 million yearsago): divergence dates are estimated 2.6–4.4 times later thanthose of the two species of Varanas and the two species ofcrocodiles (Crocodylus niloticus and Alligator mississippiensis);(2) the species Sphenodon is closely related to the group of
tortoises; (3) the primary structures of β-globin chains fromthe sea turtle Caretta and the sea snake Liophis miliaris
hemoglobins were, in particular, shown to be unusual relatedness from the group of terrestrial species in turtles andsquamates, respectively; (4) in the branches of α-globin chains
the squamates (snakes and lizards) diverged from the groupsof turtles and crocodiles, but in the branches of β-globin chains
the crocodiles first separated from the other groups of reptiles(turtles and squamates).
One of the most interesting objectives is, therefore,
determining when and how the Galapagos giant tortoises, G.
elephantopus, diversified from their sister species, the Aldabra
giant tortoises G. gigantea. At the present time, the habitatsof the two giant tortoises are remote oceanic islands and separated by two continents, Africa and South America, and theAtlantic Ocean. According to our β-globin data, the divergencetime of G. gigantea and G. carbonaria was estimated as 17
myr (million years) ago. This coincides with the recent study521

of Caccone et al (1999) who have estimated that the colonization of Madagascar by tortoises occurred in 22–14 myr ago
based on tortoises mtDNA sequences. It is not unreasonable
to make a scenario that the two living giant species had diversified less than 17 myr ago from their common ancestor andreached oceanic islands, one is volacinc (the Galapagos) andthe other is atoll (Aldabra), by rafting, the most reliable way of
migration for terrestrial animals. Future studies on globin structures of G. elephantopus may well explain the divergence times
and molecular relationships of hemoglobins among the three
Geochelone species.

ACKNOWLEDGMENTS
We thank the staff of veterinary surgeons of Animal Hospital of
Osaka Municipal Tennoji Zoo for their cooperation in collecting bloodof the Aldabra giant tortoises, G. gigantea. We also thank Dr. M. Esumi,
Department of Pathology, Nihon University School of Medicine, forher generosity in helping to prepare the equipment for analyzing aminoacid sequences.

REFERENCES
Abbasi A, Wells RMG, Brittain T, Braunitzer G (1988) Primary structure of the hemoglobins from Sphenodon (Sphenodon punctatus,
Tuatara, Rhyncocephalia). Biol Chem Hoppe-Seyler 369: 755 –764
Abbasi A, Braunitzer G (1991) Primary structure of hemoglobin from
Monitor Lizard (Varanus exanthematicus albigularis-Squamata).
Biol Chem Hoppe-Seyler 372: 473–479
Benton MJ (1990) Phylogeny of the major tetrapod groups: Morphological data and divergence dates. J Mol Evol 30: 409–424
Bonilla GO, Focesi A Jr, Bonaventura C, Bonaventura J, Cashon RE(1994) Functional properties of the hemoglobin from the SouthAmerican snake Mastigodryas bifossatus. Comp Biochem Physiol
109A: 1085 –1095
Bordin S, Meza AN, Saad STO, Ogo SH, Costa FF (1997) cDNAderived amino-acid sequence of a land turtle ( Geochelone
carbonaria) β-chain hemoglobin. Biochem Mol Biol Int 42: 255 –260
Brittain T (1988) An investigation of the functioning of the two major
haemoglobins of the Sphenodon using fast reaction kinetic methods. Biochem J 251: 771–776
Brown, JL, Ingram, VM (1974) Structural studies on chick embryonic
hemoglobins. J Biol Chem 249: 3960–3972
Bunn HF, Forget BG (1986) In Hemoglobin: Molecular, genetic andclinical aspects. Chap 2, Hemoglobin Structure, pp 13–35, Chap
3, Hemoglobin function, pp 37–60, Saunders WB Co, Philadelphia
Caccone A, Amato G, Gratry OC, Behler J, Powell JR (1999) A molecular phylogeny of four endangered Madagascar tortoises basedon mtDNA sequences. Mol Phylogenet Evol 12: 1–9
Carroll RL (1969) Origin of reptiles. In; Biology of Reptilia, Gans C etal, Ed, Vol 1, p.1– 44, Academic Press, London & New York
Dolan M, Dodgson JB, Engel JD (1983) Analysis of the adult chicken
β-globin gene. Nucleotide sequence of the locus, microheterogeneity at the 5'-end of β-globin mRNA, and aberrant nuclear
RNA species. J Biol Chem 258: 3983–3990
Duguet M, Chauvet J-P, Acher R (1974) Phylogeny of hemoglobins:
The complete amino acid sequence of an α-chain of viper (Vipera
aspis) hemoglobin. FEBS Let 47: 333–337
Felsenstein J (1993) PHYLIP: Phylogeny Inference Package, Version 3.5c. Department of Genetics SK-50, University of Wash-522

F. Shishikura and K. Takami

ington, Seattle, WA 98195, USA
Friedman M, Krull LH, Cavins JF (1970) The chromatographic determination of cystine and cysteine residues in proteins as S-β-(4pyridylethyl) cysteine. J Biol Chem 245: 3568–3871
Fushitani K, Higashiyama K, Moriyama EN, Imai K, Hosokawa K(1996) The amino acid sequences of two α chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes. Mol Biol Evol 13: 1039 –1043
Goodman M, Moore GW, Matsuda G (1975) Darwinian evolution in
the genealogy haemoglobin. Nature 253: 603– 608
Goodman M, Pedwaydon J, Czelusniak J, Suzuki T, Gotoh T, Moens
L, Shishikura F, Walz DA, Vinogradov SN (1988) An evolutionary tree for invertebrate globin sequences. J Mol Evol 27: 236 –249
Gorr TA, Mable BK, Kleinschmidt T (1998) Phylogenetic analysis of
reptilian hemoglobins: Trees, rates, and divergences. J Mol Evol
47: 471– 485
Hagopian HK, Ingram VM (1971) Developmental changes of erythropoiesis in cultured chick blastoderms. J Cell Biol 51: 440– 451
Ikehara T, Eguchi Y, Kayo S, Takei H (1997) Isolation and sequencing of two α-globin genes αA and αD in pigeon and evidence forembryo-specific expression of the αD-globin gene. Biochem
Biophys Res Commun 234: 450–453
Islam A, Persson B, Zaidi ZH, Jörnvall H (1990) Sea snake(Microcephalophis gracilis) hemoglobin: Primary structure andrelationships to other forms. J Prot Chem 9: 533–541
Jackson MH (1984) Galapagos, a natural history, University of Calgary
Press, Alberta, Canada
Jekel PA, Weijer WJ, Beintema JJ (1983) Use of endoproteinase LysC from Lysobacter enzymogenes in protein sequence analysis.
Anal Biochem 134: 347–354
Keilin D (1956) Distribution and diversity of haemoglobin. Acta Biochim
Polon 3: 439–457
Kleinschmidt T, Sgouros JG (1987) Hemoglobin sequences. Biol Chem
Hoppe-Seyler, 368: 579-615
Knöchel W, Meyerhof W, Hummel S, Grundmann U (1983) Molecularcloning and sequencing of mRNAs coding for minor adult globin
polypeptides of Xenopus laevis. Nucl Acids Res 11: 1543–1553
Leclercq F, Schnek AG, Braunitzer G, Stangl A, Schrank B (1981)
Direct reciprocal allosteric interaction of oxygen and hydorgencarbonate sequence of the haemoglobins of the caiman (Caimancrocodylus), the Nile crocodile (Crocodylus niloticus) and theMississippi crocodile (Alligator mississippiensis). Hoppe-Seyler’s
Z Physiol Chem 362; 1151–1158
Leclercq F, Stangl A, Schrank B, Vandecasserie C, Paul C, Schnek
AG, Leonis J, Braunitzer G (1982) Caiman crocodylus hemoglobin. Complete primary structures of α and β chains; phylogenic
and regulatory aspects. Int J Peptide Protein Res 20: 337–350
Liu C-S (1975) Preparation and chemical characterization of the threechains of the major hemoglobin of the sea snake, Pelamis
platurus. J Biochem 78: 19–29
Matsuura MSA, Fushitani K, Riggs AF (1989) The amino acidsequences of the α and β chains of hemoglobin from the snake,
Liophis miliaris. J Biol Chem 264: 5515– 5521

Naqvi S, Zaidi ZH, von Bahr-Lindström H, Carlquist M, Jörnvall H(1983) Characterization of hemoglobin from the lizard Uromastix
hardwickii. FEBS Lett 162: 290–295
Naqvi S, Abbasi A, Zaidi ZH (1994) Primary structure of hemoglobinfrom cobra Naja naja naja. J Prot Chem 13: 669– 679
Oberthür W, Braunitzer G, Baumann R, Wright PG (1983) Die
Primärstruktur der α- und β-Ketten der Hauptokomponentender Hämoglobine des Straußes (Struthio camelus) unddes Nandus (Rhea americana) (Struthioformes). Aspekte zur
Atmungsphysiologie und Systematik. Hope-Seyler’s Z Physiol
Chem 364: 119 –134
Oberthür W, Godovac-Zimmermann J, Braunitzer G (1986) The
expression of αD-chains in the hemoglobin of adult ostrich (Struthio
camelus) and American rhea (Rhea americana). The different
evolution of adult bird αA-, αD- and β-chains. Biol Chem HoppeSeyler 367: 507–514
Patient RK, Harris R, Walmsley ME, Williams JG (1983) The complete nucleotide sequence of the major adult β globin gene of
Xenopus laevis. J Biol Chem 258: 8521–8523
Petruzzelli R, Aureli G, Lania A, Galtieri A, Desideri A, Giardina B(1996) Diving behaviour and haemoglobin function: the primarystructure of the α- and β-chains of the sea turtle (Caretta caretta)
and its functional implications. Biochem J 316: 959–965
Rücknagel KP, Reischl E, Braunitzer G (1984) Hämoglobine der Reptilian. Expression von αD-Genen bei Schildkröten, Chrysemys
picta bellii und Phrynops hilarii (Testudines). Hoppe-Seyler’s Z
Physiol Chem 365: 1163–1171
Rücknagel KP, Braunitzer G, Wiesner H (1988) Hemoglobins of reptiles. The primary structures of the αI- and βI-chains of common
iguana (Iguana iguana). Biol Chem Hoppe-Seyler 369: 1143–
1150
Rücknagel KP, Braunitzer G (1988) Hemoglobins of reptiles. The primary structure of the major and minor hemoglobin component of
adult Western Painted Turtle (Chrysemys picta bellii). Biol Chem
Hoppe-Seyler 369: 123–131
Shishikura F, Snow JW, Gotoh T, Vinogradov SN, Walz DA (1987)
Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris. J Biol Chem 262: 3123–
3131
Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving
the sensitivity of progressive multiple sequence alignment throughsequences weighting, positions-specific gap penalties and weightmatrix choice. Nucleic Acid Res 22: 4673– 4680
Vinogradov SN, Walz DA, Pohajdak B, Moens L, Kapp OH, Suzuki T,
Trotman, CNA (1993) Adventitious variability? The amino acidsequences of nonvertebrate globins. Comp Biochem Physiol
106B: 1–26
Weber RE, Kleinschmidt T, Abbasi A, Wells RMG, Braunitzer G(1989) Allosteric transition in hemoglobin (α A2βI2) from the
Phynchocephalian reptile relict Sphenodon punctatus. Hemoglobin 13: 625–636(Received December 3, 2000 / Accepted January 22, 2001)

Primary Structures of G. gigantea globins
Appendix 1. Sequence analyses of peptides obtained by cleavage with lysyl endopeptidase and V8 protease.

523

524

F. Shishikura and K. Takami

Primary Structures of G. gigantea globins

525

526

F. Shishikura and K. Takami

Appendix 2. Alignment of amino acid sequences of 28 reptilian globins.
Clustal W (Thompson et al. 1994), a multiple alignment program, was used. The invariant amino acid residues are indicated by asterisks. The
nomenclatures of globin-chains with a numerical order are the same as Fig. 4.

Micron 35 (2004) 123–125
www.elsevier.com/locate/micron

Leech hemoglobin: primary structures of four kinds of globinsfrom Haemadipsa zeylanica var. japonica
Fumio Shishikura*
Biology Department, Nihon University School of Medicine, Tokyo 173-8610, Japan

1. Introduction
The extracellular hemoglobin of the land leech Haemadipsa zeylanica var. japonica is basically composed of threeconstituent subunits, a dimer (D1-globin and D2-globin)
and two monomers (M1-globin and M2-globin) (Shishikura
et al., 1997). They were separated into two distinct groups,
A and B, originally proposed by Gotoh et al. (1987) for themultisubunit globins of annelids. Our previous report on theamino (N)-terminal amino acid sequences of the leech
globins has shown that D1-globin and M1-globin belong togroup A, and D2-globin and M2-globin to group B(Shishikura et al., 1997).
As for the complete primary structure of hemoglobin ofannelids, many of studies have been performed onoligochaetes and polychaetes. However, to date, there arefew studies on leech hemoglobin. To establish the primarystructures of the leech globins is therefore required, and theirstructures should be useful in clarifying the molecular
evolution of hemoglobin in annelids and other invertebrates,
as well as in vertebrates. Here the author reports
the complete primary structures of the four globins fromthe land leech H. zeylanica by nucleotide and peptide
sequencing.

2. Materials and methods
2.1. Preparation of four kinds of globins
and globin-chain separation
Globins of H. zeylanica were prepared by the method
described previously (Shishikura et al., 1997). One dimer
subunit and two monomer subunits were separated by
gel-ﬁltration on a Superdex 75 column. To separate the D1globin and D2-globin from the dimer, the disulﬁde-bonds of* Fax: þ81-2-3972-0027.
E-mail address: fshishi@med.nihon-u.ac.jp (F. Shishikura).
0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2003.10.040

the dimer subunit were cleaved by reduction andS-pyridylethylation (Friedman et al., 1970), followedby isolation of each globin-chain on a Resource RPC
column.
2.2. Protein sequencing
All four globin-molecules modiﬁed by S-pyridylethylation were digested, separately, with Lysyl endopeptidase (Wako Pure Chemicals Co., Tokyo). Peptide fragments
derived from each of the parent molecules were separatedusing a reversed-phase column, Resource. Sequence
analyses of these fragments were performed using a gas
phase protein sequencer, PPSQ-10 (Shimadzu Co., Kyoto),
equipped with a class LC-10 amino acid analyser.
Phenylthiohydantoin (PTH)-derivatives from the sequencer
were separated and quantiﬁed. The peptides were thenaligned with the assistance of sequence similarities ofknown globin structures of annelids.
2.3. Nucleotide sequencing
Total RNA from H. zeylanica was extracted from thewhole body by acid guanidium thiocyanate – phenol –chloroform method (Chomczynski and Sacchi, 1987), andmRNAs including the four kinds of globins were isolatedwith a TaKaRa Oligotexe-dT30 , Super . mRNAPuriﬁcation Kit (TaKaRa Bio Inc., Shiga). The singlestranded cDNAs were synthesized with a TaKaRa RNA
PCR Kit (v. 2.1) using the Oligo dT-Adaptor Primer (M13primer M4, 17-mer).
For PCR ampliﬁcation of the 30 region of the cDNA, theprimers used were the adaptor and a redundant oligomer
based on N-terminal amino acid sequence of each globin.
The second PCR-ampliﬁcation was conducted with a nested
PCR primer (redundant oligomer) and the adaptor. One
major fragment was detected on agarose-gel electrophoresis. Then, the fragment was puriﬁed and sequenced directlywith a BigDye Terminator v1.1 Cycle Sequencing Kit

124

F. Shishikura / Micron 35 (2004) 123–125

Fig. 1. Alignment of primary structures of the four globins from H. zeylanica hemoglobin. Accession numbers of gene banks ((DDBJ/EMBL/Genebank) are: M1globin, AB119122; M2-globin, AB119123; D1-globin, AB119124; D2-globin, AB119125. p indicates positions which have a single, fully conserved residue.

(Applied Biosystems, Foster City, CA). The rest of the 30
end was afterwards conﬁrmed by 30 RACE with the adaptor
and a gene-speciﬁc primer. All the forward and reverse
primers, except for the Oligo dT, were tagged with
pUC/M13 sequencing forward 17-mer (Sigma-Aldrich
Japan, Tokyo) and reverse 17-mer (Promega, Tokyo),respectively.
For PCR ampliﬁcation of the 50 region of the cDNA,
gene-speciﬁc primers with or without 50 mophosphate were
designed in order to extend the sequence in the 50 end usinga TaKaRa 50 -Full RACE Core Set (Maruyama et al., 1995).

Fig. 2 shows a phylogenetic tree based on globin primarystructures of Hirudinea, Oligochaeta, and Polychaeta. Threeclusters can be seen: strain A, strain B, and others including
M2-globin (B type globin) and Lumbricus III globin (B typeglobin). The present tree strongly indicates that M2-globin
is a type of ancestral globins in annelids.
In conclusion, this study has provided the primarystructures of the four globins from the land leech
H. zeylanica, thereby allowing the construction of acomparative molecular phylogenetic tree of the globins of

2.4. Computer analysis
A multiple alignment program, Clustal X (Jeanmougin
and Thompson, 1998), was used in the alignment of the four
H. zeylanica globin chains, including those of representative
oligochaete and polychaete species. A phylogenetic tree wasalso constructed by neighbor-joining method stored in the
program.

3. Results and discussion
Two sequencing methods, protein and nucleotide sequencing, provided sufﬁcient information to establish thecomplete primary structures of the four H. zeylanica globins.
Fig. 1 shows the complete primary structures of these fourglobin chains, whose sequences are reinforced by thedifferent method (nucleotide sequences are not shownhere). The mature globin-molecules are composed of 146
amino acid residues for M1-globin, 156 for M2-globin, 143
for D1-globin, and 149 for D2-globin. There are 22 invariant
amino acids in the alignment. It is noticeable that the aminoacid replacements occur at positions 57 (phenylalanine toleucine in D2-globin), 73 (histidine to phenylalanine in
D1-globin), 88 (leucine to phenylalanine in D2-globin), and
139 (alanine to serine in M1-globin), as shown in Fig. 1.
When compared sequence similarities among the fourglobins, 26 –33% identities are found.

Fig. 2. Phylogenetic tree, based on globin-primary structures ofrepresentative species of Annelida, constructed from 1000 bootstrap
replications by the neighbor-joining method. Primary structures ofTylorrhynchus I, IIA, IIB, and IIC were quoted from Suzuki and Gotoh(1986). Primary structures of Lumbricus I and the other three globins of
Lumbricus (II, III, and IV) were taken from Shishikura et al. (1987) andFushitani et al. (1988), respectively.

F. Shishikura / Micron 35 (2004) 123–125

representative species belonging to three orders of Annnelida (Hirudinea, Oligochaeta, and Polychaeta). From thestructure of the land leech globins, Hirudinea have ancestralcharacteristics among annelids.

Acknowledgements
The author thanks Dr S. Tani and Mr Y. Shishikura forcollecting and providing H. zeylanica. This work was
supported in part by a Grant from the Ministry of Education,
Culture, Sports, Science and Technology to promote multidisciplinary research projects.

References
Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolationby acid guanidinium thiocyanate –phenol–chloroform extraction. Anal.
Biochem. 162, 156–159.

125

Friedman, M., Kruill, L.H., Cavins, J.F., 1970. The chromatographic
determination of cystine and cysteine residues in proteins as S-b(4-pyridylethyl)cysteine. J. Biol. Chem. 245, 3868 – 3871.
Fushitani, K., Matsuura, M.S.A., Riggs, A.F., 1988. The amino acid sequencesof chains a, b and c that form the trimer subunit of the extracellularhemoglobin from Lumbricus terrestris. J. Biol. Chem. 263, 6502–6517.
Gotoh, T., Shishikura, F., Snow, J.W., Ereifej, K.I., Vinogradov, S.N.,
Walz, D.A., 1987. Two globin strains in the giant annelid extracellular
haemoglobins. Biochem. J. 241, 441–445.
Jeanmougin, F., Thompson, J.D., 1998. Multiple sequence alignment with
Clustal X. Trends Biochem. Sci. 23, 403–405.
Maruyama, I.N., Rakow, T.L., Maruyama, H.I., 1995. cRACE: a simple
method for identiﬁcation of the 50 end of mRNAs. Nucleic Acids Res.
23, 3796–3797.
Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N., Walz, D.A., 1987.
Amino acid sequence of the monomer subunit of the extracellularhemoglobin of Lumbricus terrestris. J. Biol. Chem. 262, 3123–3131.
Shishikura, F., Ochiai, T., Yamanaka, I., 1997. Leech extracellularhemoglobin: two globin strains that are akin to vertebrate hemoglobina and b chains. Zool. Sci. 14, 923–930.
Suzuki, T., Gotoh, T., 1986. The complete amino acid sequence of giant
multisubunit hemoglobin from the polychaete Tylorrhynchus heterochaetus. J. Biol. Chem. 261, 9257–9267.

Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

The complete amino acid sequences of four globins from the land
leech Haemadipsa zeylanica var. japonica
Fumio Shishikura*
Biology Department, Nihon University School of Medicine, 30-1, Oyaguchi-kami-machi, Itabash, Tokyo 173-8610, Japan
Received 17 October 2003; received in revised form 15 January 2004; accepted 17 January 2004

Abstract
The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determinedusing nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acidresidues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kindsof globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking featureof amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine isconserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus andTylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures
including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T.
heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), andhuman a and b globins strongly indicated that the leech globins first separated from globin lineage of annelids.
䊚 2004 Elsevier Inc. All rights reserved.
Keywords: Annelida; c-DNA; Evolution; Globin; Haemadipsa zeylanica; Nucleotide sequence; Primary structure; RACE

1. Introduction
Although leeches occur in habitats ranging fromaquatic (both freshwater and marine) to terrestrial
ecosystems and are found on all continents, thesystematics of leeches is poorly understood (Mann,
1962; Apakupakul et al., 1999). The jawed Japanese land leech (Haemadipsa zeylanica var. japonica) is known to have a sanguivorous habit andbelongs to the Hirudiniformes, which includes the
medicinal leech family Hirudinidae and the terrestrial Haemadipsidae. Recently, the salivary components of sanguivorous leeches have been
investigated for pharmaceutical and clinical uses
*Corresponding author. Tel.: q81-3-3972-8111x2291; fax:
q81-3-3972-0027.
E-mail address: fshishi@med.nihon-u.ac.jp (F. Shishikura).

in the prevention of blood clot formation (Walsmann and Markwardt, 1985; Lent, 1986).
The extracellular hemoglobin of the land leech,
H. zeylanica var. japonica, is basically composedof three constituent subunits, a dimer (D-1 and D2 globins) and two monomers (M-1 and M-2
globins) (Shishikura et al., 1997). The multiple
subunit globins of the annelids were separated into
two distinct groups, strain A and strain B (Gotoh
et al., 1987). Our previous report on the first 30
amino (N)-terminal amino acid sequences of theleech globins showed that D-1 globin and M-1
globin belong to strain A, and D-2 and M-2 globins
belong to strain B (Shishikura et al., 1997).
Many studies of the primary structure of hemoglobin of the annelids have been performed inoligochaetes and polychaetes; however, to date,

1096-4959/04/$ - see front matter 䊚 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2004.01.003

444

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

few studies on leech hemoglobin have been reported (Kapp et al., 1990; Shishikura et al., 1997).
Determination of the primary structures of leech
globins, therefore, is required, and their structures
may be useful tools for clarifying the molecular
evolution of the globin super-families of annelids
in particular as well as invertebrates and vertebrates in general.
Here I report the complete primary structures ofthe four kinds of globins from the land leech, H.
zeylanica, by nucleotide sequencing and peptide
sequencing. A phylogenetic tree based on globin
structures strongly indicates that the globins of
Hirudinea first diverged from the lineage of globinsof Annelida.
2. Material and methods
2.1. Preparation of four globins and globin chain
separation
Globins of H. zeylanica were prepared by themethod described previously (Shishikura et al.,1997). The one dimer subunit and two monomer
subunits were separated by gel-filtration on a
Superdex 75 column. To separate D-1 globin and
D-2 globin, the disulfide-bonds of the dimer
subunit were irreversibly cleaved by reduction andS-pyridylethylation (Friedman et al., 1970), followed by isolation of each globin chain on a
Resource RPC column (Amersham Biosciences,
Tokyo). Globin chains were ascertained in accordance with previously determined N-terminal amino acid sequences of the four kinds of globins(Shishikura et al., 1997).
2.2. Protein sequencing
All four kinds of globin molecules modified by
reduction and S-pyridylethylation were separately
digested with Lysyl endopeptidase (Wako Pure
Chemicals, Tokyo) at an enzymeysubstrate ratioof 1y30 (molymol) for 4 h at 37 8C in 0.1 M
ammonium bicarbonate (pH 8.2). Peptide fragments derived from each of the parent molecules
were separated using a reversed-phase column(Resource RPC) in a 0.1% trifluoroacetic acid
(TFA) buffered gradient to 60% acetonitrile in
0.08% TFA. Flow rates were maintained at 0.3
mlymin. All fractions were monitored at 214 and
280 nm. Re-chromatography of selected peptides
was conducted as previously described (Shishikura

et al., 1987). Sequence analyses of these fragments
were performed using a gas phase protein sequencer, PPSQ-10 (Shimadzu, Kyoto, Japan), equippedwith a class LC-10 amino acid analyser. Phenylthiohydantoin (PTH)-derivatives from thesequencer were separated and quantified. The peptides then were aligned with the assistance ofsequence similarities toward the known globin
structures of annelids.
2.3. Nucleotide sequencing
Total RNA from H. zeylanica was extracted
from three adults (approx. 500 mg in total mass;
two to three individuals) by the acid guanidium
thiocyanate–phenol–chloroform method (Chomczynski and Sacchi, 1987), and mRNAs (approx.
450 ng in total) including the four kinds of globin
mRNAs were isolated with a Takara Oligotex娃dT30 NSuperM mRNA Purification Kit (Takara Bio,
Shiga, Japan). The single-stranded cDNAs weresynthesized with a Takara RNA PCR Kit (v. 2.1)
using the Oligo dT-Adaptor Primer (M13 primer
M4, 17-mer: 59-GTTTCCCAGTCACGACT15-39),
according to the manufacturer’s instructions.
For PCR amplification of the 39 region of thecDNA, the primers used were the adaptor and aredundant oligomer based on the N-terminal aminoacid sequence of each globin (see Appendix 2-A).
The second PCR-amplification was conducted witha nested PCR primer (a redundant oligomer) andthe adaptor (Appendix 2-B). One major fragment
was detected on agarose–gel electrophoresis ineach PCR. Then, the fragment was purified by a
GenElute娃 Agarose Spin Column (Sigma-Aldrich,
St. Louis, MO, USA), and sequenced directly witha BigDye Terminator v1.1 Cycle Sequencing Kit(Applied Biosystems, Foster City, CA, USA). The
rest of the unknown sequence of the 39 end wasafterwards confirmed by 39RACE (Frohman et al.,1988), with the adaptor and a gene-specific primer(Appendix 2-C).
For PCR amplification of the 59 region of
cDNAs, gene-specific primers with or without 59
monophosphate as listed in Appendix 2-D were
designed in order to extend the sequences in the
59 ends using a Takara 59-Full RACE Core Set(Maruyama et al., 1995), according to the manufacturer’s instructions. All forward and reverse
primers, except for the oligo dT, listed in Appendix
2, were tagged with pUCyM13 sequencing primers(forward 17-mer: 59-GTAAAACGACGGCCAGT-F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

445

39, Sigma-Aldrich Japan, Tokyo, and reverse 17mer: 59-CAGGAAACAGCTATGAC-39, Promega,
Tokyo).
2.4. Computer analysis
A multiple alignment program, Clustal X (Jeanmougin and Thompson, 1998) as well as Clustal
W (Thompson et al., 1994), was used to align thefour kinds of leech globins, with those of representative species from oligochaetes and polychaetes. Pairwise distances among 18 globin
sequences were calculated using the computer
program Protdist under the Dayhoff PAM matrix
option of the PHYLIP package (Felsenstein,1993). A phylogenetic tree was constructed by theneighbor-joining method (Saitou and Nei, 1987),
and the tree was drawn by the NJ-prot program.
3. Results and discussion
Fig. 1 shows four chromatograms of separationof peptide fragments generated from the parent
molecules, M-1 globin, M-2 globin, D-1 globin,
and D-2 globin. Fragments were selected andsequenced completely except for k-4 of D-1 globin
and k-6 of D-2 globin. As summarized in Appendix 1, the k-peptides from the four kinds of globins
were aligned with the assistance of sequence similarities towards known sequences of globins ofannelids stored in the SwissProt data bank (http:yyus.expacy.orgycgi-biny).
Using primers listed in Appendix 2, cDNAfragments amplified by PCR, whose major bands
were extracted from the agarose gels andsequenced, are shown in Fig. 2. Results of nucleotide sequencing of 12 cDNA-fragments provided
enough information to determine complete cDNA
sequences. Entire coding regions of the four kindsof H. zeylanica globins have been stored in
GenBankyDDBJyEMBL(accessionnos.
AB119122 for M-1 globin, AB119123 for M-2
globin, AB119124 for D-1 globin, and AB119125
for D-2 globin). The nucleotide sequences ofmature proteins, as shown in Appendix 1, arecomprised of 146 amino acid residues for M-1
globin, 156 for M-2 globin, 143 for D-1 globin,
and 149 for D-2 globin. The amino acid sequences
deduced from the nucleotide sequences were identical with those determined by protein analyses
described above. Their sequences reinforce eachother. In addition, protein analyses demonstrated

Fig. 1. Separation profiles of peptide fragments by reversedphase column chromatography in FPLC. (A) M-1 globin, (B)M-2 globin, (C) D-1 globin, (D) D-2 globin. After digestionof the parent molecules (approx. 200 mg) with lysyl endopeptidase, each sample was applied to a Resource RPC column (3ml packed with Source 15 RPC). All peaks with numbers weresequenced, and their amino acid sequences are shown inAppendix 1.

two kinds of k-peptides (asterisks in Fig. 1), ineach of which a micro heterogeneity of aminoacids was detected, while by nucleotide analyses
no trace of nucleotides indicating micro heterogeneity was found.
Fig. 3 shows an alignment of the four globins,
in which there are 22 invariant amino acids (asterisks). Among them, 11 amino acids are common(dots) when compared with Lumbricus globins
and Tylorrhynchus globins (sequences not shown).
When they were aligned with human b globin,

446

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

Fig. 2. Agarose gel electrophoreses (1.5% gels) of the PCR products amplified from four kinds of cDNAs using primers as listed inAppendix 2. (A) Nested PCR, (B) 39-RACE, (C-1) 1st PCR of 59-RACE, (C-2) 2nd PCR of 59-RACE. The major fragment in eachlane, except for the S1 and S2 lanes, was extracted from the gel and sequenced. PCR conditions: 30 cycles each consisting of 30 s at
94 8C for denaturation, 30 s at 50 8C for annealing, and 1 min at 72 8C for primer extension. S1 (100 bp ladder) and S2 (pHY) are
DNA markers.

Fig. 3. Alignment of the amino acid sequences of the four kinds of globins from H. zeylanica. The 22 amino acid residues conservedin the four kinds of globins are shown by asterisks. Residue No. is an arbitrary residue number with the numbering beginning fromthe N-terminal of the globins with the longest N-terminal extension. ‘Helix position’ refers to the helix position in human b globin(Nagel, 1995).

447

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

only six amino acids were invariant (shown by
open circles): A12-Trp, B16-Val, EF5-Leu, F4Leu, F8-His and G16-Leu.
Four amino acid substitutions, underlined in Fig.
3, are noticeable because they occurred in only
one of the four kinds of globin-chains of H.
zeylanica compared with Lumbricus globins andTylorrhynchus globins: a Leu at position 57 of the
D-2 globin, a Phe at position 73 of the D-1 globin,a Phe at position 88 of the D-2 globin, and a Ser
at position 139 of the M-1 globin. Among them,
the alteration of the Phe at position 73 of the D-1
globin is, in particular, noteworthy because thecorresponding His at helix position E7 (E7-His),
known as the distal His, is conserved widely invertebrates and invertebrates. Phillips and Schoenborn (1981) reported that the function of substituteresidues (E7-Gln or E7-Leu) are not clear,although they do present steric hindrance to linear
ligands, such as carbon monoxide, and favor ‘bent’ones, such as O2. Nagai et al. (1987) produced amutant human hemoglobin with E7-Gln, Val or
Gly using protein engineering and showed that thesteric hindrance of ligand binding by the E11

residue and the polarity of the E7 residue in the bsubunit were critical for fine-tuning ligand affinity.
They also showed that E7-His and E7-Gln areboth capable of donating a hydrogen bond to theoxygen molecules in a similar manner and the E7His to Gln substitution is therefore a structurally
and functionally conservative change. A Leu substitution at an E7-His position was also found ina Glycera globin (Imamura et al., 1972). In thisstudy I found a Phe substitution at the E7-His
position in one of the four kinds of globins of
Haemadipsa (D-1 globin). Most recently, Suzuki
and Vinogradov have reported a Phe substitution
at position E7 in a fresh water leech globin(Macrobdella IIA, GenBank accession no.
AB118638). Suzuki et al. (1989b) found that
hemoglobin of the deep sea clam, Calyptogena
soyoae, (E7-Gln, Suzuki et al., 1989a) was autoxidized 1300 times faster than human hemoglobin
under the same conditions. Since the pioneering
work of Perutz (1970) who first studied geometriesof distal residues in the oxygen binding sites of
myoglobin and hemoglobin, E7 substitutions of
the conserved His residue remain an interesting

Table 1
Percent identities and pair wise distances between Haemadipsa globins, Macrobdella globins, Lumbircus globins, Tylorrhynchus globins,
and the human a and b-globin
GlobinH. zeylanica
M1

M1
M2

M2

D1

D2

IIA

26.11
(2.089)32.88(1.783)30.57(1.567)30.46(1.985)30.77(1.687)31.33(1.596)

29.63(1.782)29.94(1.608)75.00(0.266)28.74(1.636)

D1
D2
Globin

M1
M2
D1
D2

M. decora
1

L. terrestris

IIB

2

30.54
(1.967)28.31(1.852)30.54(1.492)82.88(0.219)

B3

C4

73.17
(0.218)26.04(2.272)30.49(1.690)27.81(1.879)27.81(1.884)52.98(0.627)30.18(1.609)32.14(1.535)
Homo sapiens

T. heterochaetus

I (d)5II(b)6

III(c)6

IV(a)6

I7

IIA7

IIB7

IIC7

a8

b8

36.99
(1.420)24.84(2.003)34.03(1.520)26.00(1.910)35.62(1.514)22.29(2.178)39.31(1.186)26.00(1.926)31.17(1.975)36.94(1.233)34.42(1.426)35.95(1.472)29.61(1.850)32.69(1.477)32.90(1.380)38.16(1.303)35.37(1.450)26.92(1.723)28.47(1.690)24.50(1.905)31.51(1.720)23.57(2.456)34.93(1.426)27.81(1.814)29.33(1.885)33.33(1.331)32.90(1.467)32.89(1.335)27.15(2.205)32.05(1.738)31.33(1.544)37.58(1.240)17.22(3.245)15.72(2.980)16.45(3.513)15.79(3.651)

16.13(3.335)16.15(3.096)17.45(3.353)14.84(3.477)

Values in parentheses are genetic distances calculated by the program Protdist uder the Dayhoff PAM matrix option (Felsenstein,1993). (1) DDBJ accession no.: AB118638, (2) DDBJ accession no.: AB 118639, (3) DDBJ accession no.: AB118640, (4) DDBJ
accession no.: AB118641, (5) Shishikura et al., 1987, (6) Fushitani et al., 1988, (7) Suzuki and Gotoh, 1986, (8) Braunitzer et al.,
1961.

448

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

Fig. 4. Phylogenetic tree based on primary structures of 16 globins of Annelida, constructed from 1000 bootstrap replications by theneighbor-joining method (Saitou and Nei, 1987). The scale shown in the upper segment of the tree represents the evolutionary distances,
given as the average number of substitutions per site. The scores (with underlines) shown at each node represent bootstrap values.

objective. We are currently making crystals fromthe leech hemoglobin as well as from C. soyoae
hemoglobin.
It is well known that homologous subunits
sharing an orthologous relationship in macromolecules comprised of multiple subunits, namely,
hemoglobin and hemocyanin, are useful tools foranalyzing the genetic relationships of invertebrates
and vertebrates. I compared the primary structuresof two sets of four kinds of globins from Haemadipsa and Macrobdella (GenBank accession nos.
AB118638, AB118639, AB118640, AB118641).
Table 1 lists the percentage identities and genetic
distances between the complete amino acidsequences of Haemadipsa, and the scores werecompared with those of Macrobdella, Lumbricus,

and Tylorrhynchus as well as human a and b
globins. Low similarities (22–38%) were foundwhen leech globins were compared with Lumbricus
globins and Tylorrhynchus globins, as well as
human a globin (16–17%) and b globin (15–18%). However, very high scores (53–83%) were
found between two globins of the leeches such as
the M-1 globin vs. Macrobdella B globin (73.2%),
M-2 globin vs. Macrobdella C globin (53.0%), D1 globin vs. Macrobdella IIA globin (75.0%), andD-2 globin vs. Macrobdella IIB globin (82.9%),
suggesting each of these two globins to be inorthologous relationships. Traditionally, H. zeylanica belongs to Haemadipsidae and M. decora
belongs to Hirudinidae. The suborder of the two
species is Hirudiniformes. Siddall and Burreson

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454(1998) have reported a molecular phylogeny ofleeches, including the Haemadipsidae and Hirudinidae, based on the mitochondrial cytochrome coxidase subunit I, and confirmed a close relationship to each other in the traditional phylogeny ofleeches (Mann, 1962). This is also supported bythis study.
Gotoh and his collaborators (Gotoh et al., 1987)
proposed two strains of globins, A and B, for theclassification of multiple globins of annelids. The
two-strain hypothesis has been revised: strain A
was subdivided into A1 and A2 and strain B into
B1 and B2 (Gotoh et al., 1991; Suzuki et al.,1993). Recently, Negrisolo et al. (2001) proposeda new model for globin evolution of annelids,
vestimentiferans, and pogonophorans. They also
pointed out that the subdivision into four homologous groups of globins (A1, A2, B1 and B2)
could be an oversimplification of the real situation.
This proposal is supported by the molecular relationships of the four groups appeared on Fig. 4.
Fig. 4 shows the phylogenetic relationship ofannelid globins based on primary structures,
including a land leech (H. zylanica) and a fresh
water leech (M. decora), a terrestrial earthworm(L. terrestris; a representative species of Oligochaeta), and a marine polychaete (T. heterochaetus; a representative species of Polychaeta). In a
preceding paper (Shishikura et al., 1997), weclassified the four kinds of globins of H. zeylanica
into two strains, A and B, based on N-terminal
sequences, and this classification was confirmedby this study. Fig. 4 and Table 1 clearly support

449

four subdivisions in the cases of leech globins.
The tree also partly supports Negrisolo’s recommendation that to classify some Lumbricus globins
and Tylorrhynchus globins into four subdivisions
is an oversimplification. More information on the
primary structures is needed to establish the realmolecular relationships among leech globins in
Hirudinea because the two kinds of leeches reported here seem to have diverged in very recent times(Siddall and Burreson, 1998; Apakupakul et al.,1999).
In conclusion, this study determined the primarystructures of the four kinds of globins from theland leech, H. zeylanica, allowing the constructionof a molecular phylogenetic tree of globins among
representative species belonging to the three ordersof Annelida (Hirudinea, Oligochaeta and Polychaeta). Judging from the molecular relationshipsof annelid globins, the leech globins first separated
from the lineage of Annelida.
Acknowledgments
The author thanks Dr S. Tani and Mr Y. Shishikura for collecting and providing H. zeylanica.
This work was supported in part by a Grant from
the Ministry of Education, Culture, Sports, Scienceand Technology to promote multi-disciplinary
research projects.
Appendix A:
Appendix B:

450

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

451

452

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

453

454

F. Shishikura / Comparative Biochemistry and Physiology Part B 137 (2004) 443–454

References
Apakupakul, K., Siddall, M.E., Burreson, E.M., 1999. Higher
level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences. Mol. Phy.
Evol. 12, 350–359.
Braunitzer, G., Gehring-Muller, R., Hilschmann, N., Hilse, K.,
Hobom, G., Rudloff, V., et al., 1961. Die Konstitution desnormalen adulten Humanhamoglobins. Hoppe-Seyler’s Z.
Physiol. Chem. 325, 283–286.
Chomczynski, P., Sacchi, N., 1987. Single-step method of
RNA isolation by acid guanidinium thiocyanate–phenol–
chloroform extraction. Anal. Biochem. 162, 156–159.
Felsenstein, J., 1993. PHYLIP: Phylogen inference Package,
Version 3.5c. Department of Genetics SK-50. University ofWashington, Seattle, WA.
Friedman, M., Kruill, L.H., Cavins, J.F., 1970. The chromatographic determination of cystine and cysteine residues inproteins as S-b-(4-pyridylethyl)cysteine. J. Biol. Chem. 245,
3868–3871.
Frohman, M.A., Dush, M.K., Martin, G.R., 1988. Rapid
production of full-length cDNAs from rare transcripts:
Amplification using a single gene-sepecific oligonucleotide
primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.
Fushitani, K., Matsuura, M.S.A., Riggs, A.F., 1988. The aminoacid sequences of chains a, b and c that form the trimer
subunit of the extracellular hemoglobin from Lumbricus
terrestris. J. Biol. Chem. 263, 6502–6517.
Gotoh, T., Shishikura, F., Snow, J.W., Ereifej, K.I., Vinogradov,
S.N., Walz, D.A., 1987. Two globin strains in the giant
annelid extracellular haemoglobins. Biochem. J. 241,
441–445.
Gotoh, T., Suzuki, T., Takagi, T., 1991. In: Vinogradov, S.N.,
Kapp, O.H. (Eds.), Nomenclature of the Major Constituent
Chains Common to Annelid Extracellular Hemoglobins.
Springer-Verlag, Berlin, pp. 279–283.
Imamura, T., Baldwin, T.O., Riggs, A., 1972. The amino acidsequence of the monomeric hemoglobin component fromthe bloodworm, Glycera dibranchiata. J. Biol. Chem. 247,
2785–2797.
Jeanmougin, F., Thompson, J.D., 1998. Multiple sequencealignment with Clustal X. Trends Biochem. Sci. 23,
403–405.
Kapp, O.H., Qabar, A.N., Bonner, M.C., Stern, M.S., Walz,
D.A., Schmuck, M., et al., 1990. Quaternary structure of the
giant extracellular hemoglobin of the leech Macrobdelladecora. J. Mol. Biol. 213, 141–158.
Lent, C., 1986. New medicinal and scientific uses of the leech.
Nature 323, 494.
Mann, K.H., 1962. Leeches (Hirudinea). Their Structure,
Physiology, Ecology and Embryology. Pergamon Press, New
York, pp. 201.

Maruyama, I.N., Rakow, T.L., Maruyama, H.I., 1995. cRACE:
a simple method for identification of the 59 end of mRNAs.
Nucleic Acids Res. 23, 3796–3797.
Nagai, K., Luisi, B., Shih, D., Miyazaki, G., Imai, K., Poyart,C., et al., 1987. Distal residues in the oxygen binding siteof haemoglobin studied by protein engineering. Nature 329,
858–860.
Nagel, R.L., 1995. Disorders of hemoglobin frunction andstability. In: Handin, R.I., Lux, S.E., Stossel, T.P. (Eds.),
Blood: Principles and Practice of Hematology. J.B. Lippincott, Philadelphia, pp. 1591–1644.
Negrisolo, E., Pallavicini, A., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., et al., 2001. The evolution of
extracellular hemoglobins of annelids, vestimentiferans, andpogonophorans. J. Biol. Chem. 276, 26 391–26 397.
Perutz, M.F., 1970. Stereochemistry of cooperative effects inhaemoglobin. Nature 228, 726–739.
Phillips, S.E.V., Schoenborn, B.P., 1981. Neutron diffraction
reveals oxygen–histidine hydrogen bond in oxymyoglobin.
Nature 292, 81–82.
Saitou, N., Nei, M., 1987. The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4, 406–425.
Shishikura, F., Ochiai, T., Yamanaka, I., 1997. Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin a and b chains. Zool. Sci. 14, 923–930.
Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N., Walz,
D.A., 1987. Amino acid sequence of the monomer subunitof the extracellular hemoglobin of Lumbricus terrestris. J.
Biol. Chem. 262, 3123–3131.
Siddall, M.E., Burreson, E.M., 1998. Phylogeny of leeches(Hirudinea) based on mitochondrial cytochrome c oxidasesubunit I. Mol. Phylogenet. Evol. 9, 156–162.
Suzuki, T., Gotoh, T., 1986. The complete amino acid sequenceof giant multisubunit hemoglobin from the polychaete Tylorrhynchus heterochaetus. J. Biol. Chem. 261, 9257–9267.
Suzuki, T., Takagi, T., Ohta, S., 1989a. Primary structure of adimeric haemoglobin form the deep-sea cold-seep clam
Calyptogena soyoae. Biochem. J. 260, 177–182.
Suzuki, T., Takagi, T., Ohta, S., 1989b. Amino acid sequenceof the dimeric hemoglobin (Hb I) from the deep-sea coldseep clam Calyptogena soyoae and the phylogenetic relationship with other molluscan globins. Biochim. Biophys.
Acta 993, 254–259.
Suzuki, T., Takagi, T., Ohta, S., 1993. N-terminal amino acidsequences of 440 kDa hemoglobins of the deep-sea tube
worms, Lamellibrachia sp. 1, Lamellibrachia sp. 2 andslender vestimentifera gen. Sp. 1 evolutionary relationshipwith annelid hemoglobins. Zool. Sci. 10, 141–146.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal W:
improving the sensitivity of progressive multiple sequencealignment through sequences weighting, positions-specific
gap penalties and weight matrix choice. Nucleic Acids Res.
22, 4673–4680.
Walsmann, P., Markwardt, F., 1985. On the isolation of thethrombin inhibitor Hirudin. Thromb. Res. 40, 563–569.

Biochemical and Biophysical Research Communications 315 (2004) 815–822

BBRC
www.elsevier.com/locate/ybbrc

A novel microperoxidase activity: methyl viologen-linked
nitrite reducing activity of microperoxidaseq
Kohei Suruga,a Kiriko Murakami,a Yohei Taniyama,a Toshiyuki Hama,aHirotaka Chida,a Tadashi Satoh,b Seiji Yamada,c Wataru Hakamata,d Ryu Kawachi,aYasuhiro Isogai,e Toshiyuki Nishio,a and Tadatake Okua,*baDepartment of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japanc
RIKEN, Harima Institute/SPring-8, Mikazuki-cho, Sayo, Hyogo 679-5148, Japand
Division of Organic Chemistry, National Institute of Health Sciences, Setagaya Tokyo 158-8501, Japane
RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan

Received 13 January 2004

Abstract
To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, thefragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis ofcommercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its ﬁfth ligand. The
nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the
cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acidresidues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO
2 to NO by donating one electron, the NO was completely
reduced to NHþ
4 under anaerobic condition via ferrous–NO complexes as a reaction intermediate using visible spectra and ESR
spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing
activity similar to that of mp9 in solution, and the resin retained the activity after ﬁve uses and even 1-year storage. The mp will beable to use as a substitute for nitrite reductase.
Ó 2004 Elsevier Inc. All rights reserved.
Keywords: Microperoxidase; Cytochrome c; Nitrite reducing activity; Hemoprotein; Peptide chain

In a natural nitrogen cycle, nitrite (NO2 ) is reducedby two types of nitrite reductase. First, in dissimilatory
reduction, also called denitriﬁcation, NO
2 is used as a
respiratory terminal substrate, and two types of nitrite
reductase, copper protein [1], and Cyt cd1 [2], are involved. Second, NAD(P)H–nitrite reductase (EC1.7.99.3), ferredoxin–nitrite reductase (EC 1.7.7.1), andCyt c nitrite reductase (EC 1.7.2.2), also known as assimilatory nitrite reductases, catalyze the reduction ofAbbreviations: mp, microperoxidase; Cyt c, cytochrome c; NO2),
nitrite; NO, nitric oxide; CD, circular dichroism; ESR, electron spinresonance; Mb, myoglobin; Hb, hemoglobin.
*
Corresponding author. Fax: +81-466-84-3950.
E-mail address: oku@brs.nihon-u.ac.jp (T. Oku).
q0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.01.133

þþNO
2 to ammonia (NH4 ) [3–5]. The NH4 reduced bythese assimilatory nitrite reductases is mainly used in thesynthesis of amino acids.
A heme-containing peptide microperoxidase (mp) is
prepared by the proteolytic digestion of equine heart
Cyt c. The two cysteines (Cys14 and Cys17) of mp arecovalently attached to the iron (III)-protoporphyrin IX,
and histidine (His) 18 is coordinated to heme iron. Thusfar, there are some reports about the amino acid composition and carbon monoxide (CO) reactivity of mps.
For example, Santucci et al. [6] reported that the hemecontaining undecapeptide (Val11-Glu21), also called
mp11, is prepared by the pepsin-catalyzed hydrolysis
of equine heart Cyt c, and mp11 reacts with free His
or His-composed a-helix to investigate the reaction

816

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822

of mp11 heme iron and His. As a result, the His-composed a-helix bound to the sixth position of the heme
iron of mp11, and the structural stability of the mp11
complex and His-composed a-helix was higher than thatof mp11 and free His. There are also some reports thatthe heme of mp11 reacts with CO, and the visible absorption maxima at 564 (a), 533 (b), and 413 (soret) nm
have been determined [7–9]. Ricoux et al. [10] reported
that the octapeptide mp8 (Cys14-Glu21) obtained bypeptic and tryptic digestion of equine heart Cyt c reacts
with the nitrosoalkane (RNO) produced by nitroalkane(RNO2 ) reduction, and the new absorption maxima at
415 nm (soret peak) were obtained by the formation of
the complex between mp8 and RNO. Moreover, theheme nonapeptide mp9 (Cys14-Lys22) prepared by thetrypsin-catalyzed hydrolysis of equine heart Cyt c showsa peroxidase-like activity, and its mp9 activity is approximately 1/50 that of horseradish peroxidase [11].
However, the NO
2 reducing activity of mp and the relationship between the length of the peptide chain of mp
and the NO
2 reducing activity have not yet been reported. Nitrite reductase does not come into the market,
because of low content of the enzyme in plants andmicrobes, and of requirement of a lot of time for the
puriﬁcation. We have tried to prepared a substitute fornitrite reductase by cutting of the peptide chain ofcommercial equine heart Cyt c and determine NO
2 in anaqueous solution.
In our laboratory, the structural/functional relationships of c-type Cyt have been investigated, and the
crystal structure of Cyt c6 from the red alga Porphyra
 resolution (PDB
yezoensis has been determined at 1.57 A
code: 1gdv) [12]. We reported that the conformational
stability of M58C mutant in which the sixth ligand
Met58 of P. yezoensis Cyt c6 was replaced by cysteine tobe approximately 2.2-fold that of nonmutant Cyt c6 [13].
Moreover, the NO
2 reducing activity of equine heart
Cyt c that reacts at 100 °C for 30 min is ca. ﬁvefold thatof native Cyt c, and we presumed that the NO
2 reducing
activity of Cyt c increases with heat treatment, becauseof the following intramolecular changes that occur in
Cyt c with heat denaturation: (1) unfolding of the peptide chain, (2) exposure of heme to the solvent, (3) dissociation of the sixth ligand (Met80) from heme–iron,and (4) autoxidation [14].
In this work, mp9 (C14-K22), mp22 (V11-L32), andmp65 (G1-M65) were prepared from equine heart Cyt c(104 residues) to investigate NO
2 reducing activity as anovel activity, and the relationship between the activity
and the length of the peptide chain of mp. The physicochemical properties of these mps were investigatedusing UV/visible spectra, CD spectra, and redox titration. Moreover, mp9 which showed the highest NO
2
reducing activity was immobilized in CNBr–Sepharose
and acrylamide, and its NO
2 reducing activity wasmeasured.

Materials and methods
Preparation of microperoxidase 9, 22, and 65. Mp9 and 22 wereprepared according to Plattner et al. [11] and Cheek et al. [15]. For
the preparation of mp9 and mp22, equine heart Cyt c (Wako Pure
Chemical Industries) was digested by incubation of Cyt c in 0.1 M
Tris–HCl buﬀer (pH 8.0) containing 0.5 M urea, with trypsin for
mp9 or chymotrypsin for mp22 (protein substrate:protease ratio 1:50
by mass) at 37 °C for 24 h. Mp65 was prepared by treatment of
intact equine heart Cyt c with cyanogen bromide at 20 °C for 4 h[16]. The reaction mixture was loaded on a Toyopearl HW-40F gel
ﬁltration column (Tosoh; 1.0  85 cm). The purity of mp9 and 22 waschecked using a BioCAD 700E perfusion chromatograph equipped with a Poros R2/20 column (Applied Biosystems: 4.6 
100 mm), and the purity check of mp65 was performed by tricine
SDS–PAGE.
Measurement of nitrite reducing activity. The NO
2 reducing activity
of mps was determined according to Vega [17]. The vial containing
0.675 ml of 100 mM sodium phosphate buﬀer (pH 7.0), 0.4 ml of
10 mM sodium nitrite, 0.5 ml of 3 mM methyl viologen, and 0.125 ml of
20 lM mp solution was sealed with a butyl rubber cap. After pre-incubating the mixture at 37 °C for 5 min, the reaction was initiated byadding 0.3 ml of 100 mM sodium dithionite dissolved in 50 mM sodium
bicarbonate. The reaction was conducted under anaerobic condition at
37 °C. The reaction mixture was placed in a test tube, left to stand forseveral minutes, and then vigorously shaken until complete decolorization.
NO
2 detection was performed according to Ramirez et al. [18]. ForNO
2 detection, 1.95 ml water, 1 ml of 1% sulfanilamide, 1 ml of 0.02%
N -1-naphthylethylenediamine, and 1 ml pure water were added to 50 llof the reaction mixture. After allowing the reaction mixture to stand atroom temperature for 20 min, its optical density at 540 nm was determined. One unit of activity was deﬁned as the amount of enzyme
needed to reduce 1 lmol of NO
2 per minute. The kcat value was calculated from Lineweaver–Burk plots.
NHþ
4 detection was based on the methods described by Scheiner et
al. [19]. For the NHþ
4 detection 1.9 ml water, 1 ml of 50 mg% sodium
nitroprusside in 10% phenol, 1 ml of 0.06% sodium hypochlorite
solution dissolved in 0.1 M disodium hydrogen phosphate, 0.25 M
sodium hydroxide, and 1 ml pure water were added to 100 ll of thereaction mixture. After allowing the reaction mixture to stand at room
temperature for 60 min, its optical density at 630 nm was determined.
The inhibition of NO
2 reducing activity of mps was determinedaccording to Vega [17].
Spectroscopic analysis. The visible spectra of mps were monitoredwith a Hitachi U3310 spectrophotometer using quartz cuvettes of 1.0cm path length at 25 °C. Mp concentration was determined using apyridine hemochromogen method [20]. Circular dichroism (CD)
spectra were recorded at 25 °C in 20 mM sodium phosphate buﬀer (pH7.0), with a JASCO J-700 spectropolarimeter using 0.2-cm path length
rectangular quartz cuvettes [21]. Electron spin resonance (ESR) spectrum of mp9 was recorded at cryogenic temperatures with a JOEL
ESR spectrometer, JES-FA200.
Redox titration study. The redox titrations of mps were performed
under anaerobic condition, with a continuous stream of argon, in
100 mM sodium phosphate buﬀer, pH 7.0, at 25 °C [22]. The potentials
were measured with a Horiba F-13 pH meter equipped with an ORP
electrode, and the optical spectra were monitored throughout the titration on a Hitachi U3310 spectrophotometer. The redox mediators
were used to stabilize the solution redox potential as described by
Yamada et al. [14]. The redox data were analyzed with a theoreticalcurve based on the Nernst equation (n ¼ 1): E ¼ E0 þ ðRT =nF Þlnð½ferric=½ferrousÞ [23].
Preparation of Sepharose-immobilized and acrylamide-immobilized
microperoxidase. Sepharose-immobilized mp9 and acrylamide-immobilized mp9 were prepared according to the Shin and Oshino [24]. The

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822
binding rate of mp9 to CNBr–Sepharose 4B and the rate of inclusionof mp9 in acrylamide were 99.98% and 99.78%, respectively.

Results and discussion
Physicochemical properties of microperoxidase
The UV/visible spectra of mp65 (G1-M65), mp22
(V11-L32), and mp9 (C14-K22) are shown in Fig. 1. For
mp65, its ferric forms showed visible absorption maxima
at 535 (a þ b) and 404 (soret peak) nm, and its ferrous
forms at 549 (a), 520 (b), and 415 (soret peak) nm(Fig. 1B). These absorption peaks of mp65 indicate that
mp65 has a 6-coordination low spin [25]. The absorption
peak at 695 nm that was observed in Met80 sulfur chargetransfer to heme iron disappeared in the ferric forms of
mp65. There are three His residues (His18, His26, andHis32) in equine heart Cyt c. His18 is axially coordinatedto heme iron and acts as its ﬁfth ligand [26]. Santucci
et al. [27] reported that the fragment G1-G56 was obtained by the thermolysin-catalyzed hydrolysis of equine
heart Cyt c, and this fragment has two His residues(His18 (ﬁfth ligand) and His26 or His33 (sixth ligand))817

axially bound to the heme iron. From this study, it wasconsidered that two His residues (His18 and His26 orHis33) are coordinated to heme iron in mp65. For mp22,
its ferric forms showed visible absorption maxima at
398 nm (soret peak), and its ferrous forms at 549 (a), 520(b), and 416 (soret peak) nm (Fig. 1C). This indicatesthat mp22 has a 6-coordination low spin with His18 and
His26 as axial ligands. For mp9, its ferric forms showedvisible absorption maxima at 619 (high spin markerband) and 395 (soret peak) nm, and its ferrous forms at
549 (a) and 412 (soret peak) nm; moreover, the absorption peak at 520 (b) disappeared in the ferrous forms of
mp9 (Fig. 1D). The absorption peak at 619 nm of theferric forms of mp9 was consistent with those of deoxyMb [28] and mp11 [29], which have a 5-coordination highspin with His as an axial ligand. The absorption peak at
520 (b) nm disappeared in the ferrous forms of the M80A
Cyt c mutant [25], similar to the case of mp9. From these,
it was assumed that mp9 has a 5-coordination high spinwith His18 as the ﬁfth ligand.
The physicochemical properties of mp65, mp22, andmp9 are summarized in Table 1. The isoelectric points(pI) of mp65, mp22, and mp9 were 9.52 (theoreticalvalue: 10.06), 6.02 (9.72), and 4.28 (6.99), respectively,

Fig. 1. UV/visible spectra of the ferric (solid line) and ferrous (broken line) forms of Cyt c and mps. Absorption spectra of 10 lM Cyt c and mps were
measured in 10 mM sodium phosphate buﬀer (pH 7.0) at 25 °C. (A) Cyt c; (B) mp65; (C) mp22; and (D) mp9.

818

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822

Table 1
Physicochemical properties of mps prepared from equine heart Cyt c
mp9
pI
Absorption maxima (nm)Ferric (FeIII )4.28

Ferrous (FeII )

395
619
412

E0 (mV, pH 7.0, 25 °C)
MW

549
)132
1630

mp22
6.02
398
416
520
549
)67
3065

mp65
9.52
404
535
415
520
549
)62
8900

Cyt c
10.50
409
535
415
520
550
260
12,500Fig. 2. Circular dichroism spectra of Cyt c and mps. The spectra of Cyt
c and mps in 10 mM sodium phosphate buﬀer (pH 7.0) at 25 °C wererecorded. Lane 1: Cyt c; 2: mp65; 3: mp22; and 4: mp9.

and the pI of mp9 was the same as that of mp11 [11].
The redox potentials of mp65, mp22, and mp9 were )62,
)67, and )132 mV, and were 322, 327, and 392 mVlower than that of Cyt c (260 mV), respectively. The
redox potential of mp9 was similar to that of mp11()134 mV) [30].
The secondary structures of mp65, mp22, and mp9
were examined, and the far-UV CD spectra of these mps
were measured (Fig. 2). It was found that mp9 (C14K22) has only random coil conformations, because it
has no a-helix segment of equine heart Cyt c (Figs. 2–4).
Mp22 (V11-L32) has the helix II (K22-K27) segment ofequine heart Cyt c, but its CD spectra agreed closelywith that of mp9, which has no a-helix (Figs. 2–3). Fromthese results, it was assumed that mp22 has a random
coil conformation. The CD spectra of mp9 and 22 were
almost the same as that of mp11, which has a random
coil conformation [31]. Mp65 (G1-M65) has helix I (G1A15), helix II (K22-K27) and helix III (D50-G56) segments, and part of the helix IV (E61-E69) segment.
From the CD spectra, the absorbance at 222 nm of
mp65 was ca. 1/2 that of equine heart Cyt c (Fig. 2, lane1), but the absorbance at 200 nm which corresponds to
the random coil conﬁguration increased (Fig. 2, lane 2).
This result was similar to that of the fragment G1-G56
that was prepared by the thermolysin-catalyzed hydrolysis of equine heart Cyt c [27], and it was consideredthat the a-helices of mp65 are unfolded. From the results of redox potential and CD spectra, we assumed
that the heme of mps would be exposed to solvent with
cutting of the peptide chain.
Nitrite reducing activity of microperoxidase and its
reaction mechanism
The NO
2 reducing activity of mp65, mp22, and mp9
was measured in the presence of methyl viologen anddithionite under anaerobic condition, and the NO
2 reducing activity of the mps increased with the cutting of
the peptide chain (Fig. 3). The reason for this is given in

Fig. 3. Nitrite reduction and ammonia production of Cyt c and mps. The nitrite reducing activities of 1.25 lM Cyt c and mps were measured at 37 °C
under anaerobic condition in the presence of dithionite and methyl viologen. () ferrous sulfate; (j) hemin; (n) Cyt c; (m) mp65; (s) mp22; and (d)mp9. (A) nitrite reduction; (B) ammonia production.

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822

the middle of this section. Both the NO
2 reducing activities of ferrous sulfate which composes mp, and hemin
which has no peptide chains were approximately 1/150
that of mp9 (Fig. 3). Generally known, the hemin was
insoluble in all solvents except aqueous strong base. The
kcat (s1 ) values of equine heart myoglobin (Mb) and thefragment (V17-K46, 26 residues) prepared by the lysylendopeptidase-catalyzed hydrolysis of equine heart Mb
were 0.004 and 0.018, respectively. These values weresimilar to that of Cyt c (kcat (s1 ) ¼ 0.015), but wereapproximately 1/460 and 1/100 that of mp9. In c typeCyt, the protoheme is covalently bonded via thioether tothe two cysteines (Cys14 and 17) of the peptide chain[32], but b-type hemoproteins such as Mb and hemoglobin (Hb) have no thioether linkage [33]. From these,
Table 2Kinetic study on nitrite reducing activity of Cyt c and mps

Cyt c
mp65
mp22
mp9

Km (mM)

kcat (s1 )kcat =Km (mM/s1 )

2.453
2.140
1.830
1.420

0.015
0.120
0.800
1.834

0.006
0.056
0.440
1.292

819

it was proved that the covalent bond between heme andCys may be necessary for enhancing of the NO
2 reducing activity of mp. A kinetic study of mp for determining its NO
2 reducing activity was conducted, andthe results are shown in Table 2. The kcat (s1 ) values of
mp65, 22, and 9 were 0.120, 0.800, and 1.834, respectively. The NO
2 reducing activity of the shortest nonapeptide mp9 was approximately 1/27 and 1/6 that of
the spinach nitrite reductase (kcat (s1 ) ¼ 49.650) [34]and P. yezoensis nitrite reductase (kcat (s1 ) ¼ 10.000)[35], respectively, but this activity was ca. 3.2-fold thatof the nitrite reductase from Escherichia coli (kcat (s1 ) ¼
0.570) [36] and 120-fold that of Cyt c (104 amino acidresidues).
From these results, it was assumed that the lower
molecular in prepared mps, which were water-solubleheme peptide, showed the higher NO
2 reducing activity,
because the collision probability of mps to NO
2 would
be increased with the cutting of the peptide chain. As
shown in Figs. 1B and C (see preceding section), mp65
and mp22 have a 6-coordination. On the other hand,
mp9 has a 5-coordination and the sixth position of mp9
was vacant (Fig. 1D). Consequently, the shortest

Fig. 4. (A) Nitrite reduction and ammonia production of Cyt c and mps. (B) Eﬀect of methyl viologen on nitrite reducing activity of Cyt c and mps.
(C) Eﬀect of dithionite on nitrite reducing activity of Cyt c and mps. (D) Eﬀect of methyl viologen on nitrite reducing activity of Cyt c and mps.
Methyl viologen was added to reaction vial at 30 min. (n) Cyt c; (m) mp65; (s) mp22; and (d) mp9. Solid line: nitrite reduction; Dashed line:
ammonia production.

820

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822

Fig. 5. (A) Visible spectra of ferrous mp9 reacted with nitrite. Ferrous mp9 (dashed line) was prepared by the addition of portion of dithionite underanaerobic conditions at pH 7.0. Then a portion of 1 M sodium nitrite was added (solid line). (B) Electron spin resonance spectrum of mp9 during thenitrite reducing activity. The spectrum was monitored under liquid nitrogen.

nonapeptide mp9 had the highest NO
2 reducing activity
in prepared mps.
In the NO
2 reduction by mp, NO2 was reduced toþNH4 in the presence of methyl viologen and dithionite
under anaerobic condition, and the conversion rate wasapproximately 100% (Fig. 4A). This reaction was thesame as that of the assimilatory nitrite reductases fromspinach [34], P. yezoensis [35], and E. coli [36]. When the
methyl viologen as electron carrier was absent in thereaction mixture, the NO
2 reducing activity of mp was
approximately 1/45 that of mp in the presence of methyl
viologen (Fig. 4B). When the sodium dithionite as reductant was absent in the reaction mixture, the mp did
not show the NO
2 reducing activity at all (Fig. 4C).
After the addition of methyl viologen to the reaction
þ
mixture at 30 min, the NO
2 reduction and NH4 production were rapidly proceeded (Fig. 4D).
The visible spectra of reaction products of mp9 withNO
2 were measured at pH 7.0 (Fig. 5A). The ferric mp9
did not react with NO
2 at pH 7.0 (data not shown), but
the spectral changes of the ferrous mp9 reacted withNO
2 were observed. The soret peak shifted to 410 nm(ferrous mp9–NO
2 complex) from 412 nm (ferrous mp9)
and the a-peak (549 nm) disappeared, and these spectralchanges were also recognized in the case of mp11 reacted with CO [7]. These spectral changes were probably
due to the formation of NO
2 –Fe–His coordination. The
ESR spectrum of mp9 during the NO
2 reducing activity
was measured under cryogenic temperature (Fig. 5B).
The ESR spectrum showed a hyperﬁne structure nearand at g ¼ 2, and this indicated the ferrous–NO complex [37]. From this, it was assumed that the ferrous–NO
complexes were a reaction intermediate in the NO
2reducing activity of mps.
From these results, we presumed that ferrous mps
reduced NO
2 to NO by giving one electron, and the NO
under anaerobic
was completely reduced to NHþ
4condition via ferrous–NO complexes as a reaction
intermediate. This overall reaction was a 6-electron and8-proton reduction as well as that of the nitrite reductase[34–36].

The pH dependence of the NO
2 reducing activity of
mp9 was studied. The optimum pH for NO
2 reducing
activity of mp9 was 7.0 with sodium phosphate buﬀer(Fig. 6). This value was close to those of the nitrite
reductase from spinach (pH 7.5), and P. yezoensis (pH7.5), [35].
The eﬀect of various inhibitors on the NO
2 reducing
activity of mp9 was investigated (Table 3). Potassium
cyanide (KCN) was found to be eﬀective inhibitor, andat a KCN concentration of 2 mM, the mp9 was morethan 99% inhibition. When the CO was added to thereaction mixture, CO inhibited the NO
2 reducing activity of mp9 completely. These results were similar degree to those of nitrite reductase from spinach [37,38]. A
metal chelating reagent, o-phenanthroline, and EDTA
showed no inhibition of NO
2 reducing activity of mp9.

Fig. 6. The pH proﬁle of the nitrite reduction of mp9. Each pH wasmaintained with sodium citrate (pH 4–5), sodium phosphate (pH 6–7),
Tris–chloride (pH 8), and glycine–NaOH (pH 9).

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822
Table 3
Eﬀect of some inhibitors on nitrite reducing activity of mp9
Inhibitors

Concentration (mM)

Potassium cyanide

0.02
0.2
2.0

Inhibition (%)
91.18
92.40
99.13

Carbon monoxide

Saturated

100.0

o-Phenanthroline

0.02
0.2
2.0

0.00
0.00
0.00

EDTA

0.02
0.2
2.0

0.00
0.00
1.69

821

mp9 (Fig. 7). Heme in CNBr–Sepharose-immobilized
mp9 was exposed to a solvent, but, in the case of theacrylamide-immobilized mp9, mp9 is surrounded by amatrix. It is considered that the matrix prevents its
contact with NO
2 or an electron donor [14]. Thus, it waspresumed that the NO
2 reducing activity of CNBr–
Sepharose-immobilized mp9 is higher than that of acrylamide-immobilized mp9. The residual activities of
CNBr–Sepharose and acrylamide-immobilized mp9
were 99.0% and 99.1%, respectively, even after storage
at 4 °C for 1 year. The activity of CNBr–Sepharoseimmobilized mp9 after the ﬁfth use was 99.4%, and thus
mp9 is reusable. Moreover, the immobilized mp9 also
showed the NO reducing activity in addition to NO
2reducing activity, and the activity was nearly the same asthat of soluble mp9 (data not shown).

Conclusion
In this work, we reported for the ﬁrst time that the
mps prepared by enzymatic hydrolysis of commercial
equine heart Cyt c showed the NO
2 reducing activity as
reducing
activity of mps ina novel activity. The NO2creased with the cutting of the peptide chain, and mp9
after use had the same NO
2 reducing activity as that offreshly mp9 by immobilization. This mp may thus be a
new molecule for simple applications such as the determination of NO
2 in an aqueous solution and will beable to use as a substitute for nitrite reductase.

Acknowledgments
Fig. 7. Nitrite reducing activity of immobilized mp9. (s) free mp9; (n)Sepharose-immobilized; and () acrylamide-immobilized.

These results were close to those of nitrite reductase
from spinach and green alga Chlorella fusca [37,39].
Immobilized microperoxidase and its nitrite reducing
activity, stability, and reuse
As a rule, proteins and enzymes are unstable at hightemperatures and their recovery after a reaction is difﬁcult. We attempted to improve the reusability andstability of mp9, which showed the highest NO
2 reducing activity, and prepared the mp9 immobilized in
CNBr–Sepharose or acrylamide. The NO
2 reducing
activity of CNBr–Sepharose or acrylamide-immobilized
mp9 was then measured. The NO
2 reducing activity of
CNBr–Sepharose-immobilized mp9 was fourfold that ofacrylamide-immobilized mp9, and CNBr–Sepharoseimmobilized mp9 had nearly the same activity as soluble

We are indebted to Misses Kanako Kashiwai and Mayumi Takaku,
and Messrs. Koichi Kamiuse and Osamu Tozawa, Nihon University,
for the preparation of microperoxidases, and Dr. Toshiki Masumizu,
JEOL Ltd., for measurement of the ESR spectrum. This work was
supported in part by a Grant-in-Aid for the 21 Century’s Center of
Excellence (COE) Program, for Scientiﬁc Research (C) from the
Ministry of Education, Culture, Sports, Science and Technology ofJapan, and for the Research for the Future Program (JSPSRFTF97R16001) from the Japan Society for the Promotion of Science.

References[1] T. Matsubara, Studies on denitrication. XII. Gas production fromamines and nitrite, J. Biochem. (Tokyo) 67 (1970) 229–235.
[2] P.R. Alefounder, S.J. Ferguson, The location of dissimilatory
nitrite reductase and the control of dissimilatory nitrate reductaseby oxygen in Paracoccus denitriﬁcans, Biochem. J. 192 (1980)
231–240.
[3] R.A. Lazzarini, D.E. Atkinson, A triphospyridine nucleotidespeciﬁc nitrite reductase from Escherichia coli, J. Biol. Chem. 256(1961) 9711–9717.
[4] S. Ida, Y. Morita, Puriﬁcation and general properties of spinachleaf nitrite reductase, Plant Cell Physiol. 14 (1973) 661–671.

822

K. Suruga et al. / Biochemical and Biophysical Research Communications 315 (2004) 815–822[5] O. Eisle, A. Messerschmidt, P. Stach, G.P. Bourenkov, H.D.
Bartunik, R. Huber, P.M.H. Kroneck, Structure of cytochrome cnitrite reductase, Nature 400 (1999) 476–480.
[6] R. Santucci, A. Picciau, G. Antonini, L. Campanella, A complexof microperoxidase with a synthetic peptide: structure andfunctional characterization, Biochim. Biophys. Acta 1250 (1995)
183–188.
[7] M. Laberge, A.J. Vreugdenhil, I.S. Butler, Microperoxidase-11:
molecular dynamics and Q-band excited resonance Raman of theoxidized, reduced and carbonyl forms, J. Biomol. Struct. Dyn. 15(1998) 1039–1050.
[8] V.S. Sharma, H.M. Renney, J.F. Geibel, T.G. Traylor, A new for
the determination of ligand dissociation rate constant of carboxyhemoglobin, Biochem. Biophys. Res. Commun. 66 (1975) 1301–
1306.
[9] V.S. Sharma, M.R. Schmidt, H.M. Ranney, Dissociation of CO
from carboxyhemoglobin, J. Biol. Chem. 251 (1976) 4267–4272.
[10] R. Ricoux, J.L. Boucher, D. Mansuy, J.P. Mehy, Formation ofiron (II)-nitrosoalkane complex: a new activity of microperoxidase
8, Biochem. Biophys. Res. Commun. 278 (2000) 217–223.
[11] H. Plattner, E. Wachter, P. Grobner, A heme-nonapeptide tracer
for electron microscopy, Histochemistry 53 (1977) 223–242.
[12] S. Yamada, S.-Y. Park, H. Shimizu, Y. Koshizuka, K. Kadokura,
T. Satoh, K. Suruga, M. Ogawa, Y. Isogai, T. Nishio, Y. Shiro, T.
Oku, Structure of cytochrome c6 from the red alga Porphyra
 resolution, Acta Cryst. D 56 (2000) 1577–1578.
yezoensis at 1.57 A[13] T. Satoh, A. Itoga, Y. Isogai, M. Kurihara, S. Yamada, M.
Natori, N. Suzuki, K. Suruga, R. Kawachi, M. Arahira, T.
Nishio, C. Fukazawa, T. Oku, Increasing the conformational
stability by replacement of heme axial ligand in c-type cytochrome, FEBS Lett. 531 (2002) 543–547.
[14] S. Yamada, K. Suruga, M. Ogawa, T. Hama, T. Satoh, R.
Kawachi, T. Nishio, T. Oku, Appearance of nitrite reducing
activity of cytochrome c upon heat denaturation, Biosci. Biotechnol. Biochem. 66 (10) (2002) 2044–2051.
[15] J. Cheek, D.W. Low, H.B. Gray, J.H. Dawson, Histidine-tailed
microperoxidase-10: a pH-dependent ligand switch, Biochem.
Biophys. Res. Commun. 253 (1998) 195–198.
[16] M. Hoshino, Y. Kawata, Y. Goto, Interaction of GroEL with
conformational states of horse heart cytochrome c, J. Mol. Biol.
262 (1996) 575–587.
[17] J.M. Vega, J. Cardenas, M. Losada, Ferredoxin–nitrite reductase,
Methods Enzymol. 69 (1980) 255–270.
[18] J. Ramirez, F.F.D. Campo, A. Paneque, M. Losada, Ferredoxin–
nitrite reductase from spinach, Biochim. Biophys. Acta 118 (1966)
58–71.
[19] D. Scheiner, Determination of ammonia and Kjeldahl nitrogen byindophenol method, Water Res. 10 (1976) 31–36.
[20] D.L. Drabkin, Spectrophotometric studies, J. Biol. Chem. 146(1942) 605–617.
[21] C.N. Pace, B.A. Shirly, J.A. Thompson, Protein Structure: A
Practical Approach, IRL, Oxford, 1989, pp. 311–330.
[22] S. Yamada, T. Nakamura, Y. Tanaka, Y. Isogai, T. Nishio, T.
Oku, Characterization and amino acid sequences of cytochrome c6

from two strains of the green alga Chlorella vulgaris, Biosci.
Biotechnol. Biochem. 64 (2000) 628–632.
[23] P.L. Duton, Redox potentiometry: determination of midpoint
potentials of oxidation-reduction components of biological electron-transfer systems, Methods Enzymol. 54 (1978) 411–435.
[24] M. Shin, R. Oshino, Ferredoxin–Sepharose 4B as a tool for the
puriﬁcation of ferredoxin-NADPþ reductase, J. Biochem. (Tokyo)
83 (1978) 357–361.
[25] J.M. Wallace, I. Cardenes, Functional role of heme ligation incytochrome c, J. Biol. Chem. 267 (1992) 3852–3861.
[26] G.W. Bushnell, G.V. Louie, G.D. Brayer, High-resolution threedimensional structure of horse heart cytochrome c, J. Mol. Biol.
20 (1990) 585–595.
[27] R. Santucci, L. Fiorucci, F. Sinibaldi, F. Polizio, A. Desideri, F.
Ascoli, The heme-containing N-fragment (residues 1–56) of
cytochrome c is a bis-histidine functional system, Arch. Biochem.
Biophys. 379 (2000) 331–336.
[28] T. Iizuka, T. Yonetani, Spin changes in hemoproteins, Adv.
Biophys. 1 (1970) 157–182.
[29] H.M. Marques, C.B. Perry, Hemepeptide models for hemoproteins: the behavior of N -acetylmicroperoxidase-11 in aqueous
solution, J. Inorg. Biochem. 75 (1999) 281–291.
[30] G. Battistuzzi, M. Borsari, J.A. Cowan, A. Ranieri, M. Sola,
Control of cytochrome c redox potential: axial ligation andprotein environment eﬀects, J. Am. Chem. Soc. 124 (2002) 5315–
5324.
[31] W. Huang, Z. Zheling, X. Han, J. Tang, Z. Peng, S. Dong, E.
Wang, Electrochemistry and spectroscopy study on the interactionof microperoxidase-11 with lipid membrane, Biophys. Chem. 94(2001) 165–173.
[32] E. Margoliash, E. Smith, G. Kreil, H. Tuppy, The completeamino-acid sequence, Nature 192 (1961) 1125–1127.
[33] P.A. Cornelius, A.W. Steele, D.A. Chernoﬀ, R.M. Hochstrasser,
Diﬀerent dissociation pathways and observation of an excited
deoxy state in picosecond photolysis of oxy-and carboxymyoglobin, Proc. Natl. Acad. Sci. USA 78 (1981) 7526–7529.
[34] C.-H. Ho, G. Tamura, Puriﬁcation and properties of nitrite
reductase from spinach leaves, Agric. Biol. Chem. 37 (1973) 37–
44.
[35] M.G. Guerreo, J.M. Vega, M. Losada, The assimilatory nitritereducing system and its regulation, Ann. Rev. Plant Physiol. 32(1981) 169–204.
[36] J.D. Kemp, D.E. Atkinson, Nitrite reductase of Escherichia coli
speciﬁc for reduced nicotinamide adenine dinucleotide, J. Bacteriol. 92 (1966) 628–634.
[37] M. Hirasawasoga, S. Horie, G. Tamura, Further characterizationof ferredoxin nitrite reductase and the relationship between the
enzyme and methyl viologen-dependent nitrite reductase, Agric.
Biol. Chem. 46 (1982) 1319–1328.
[38] J.M. Vega, H. Kamin, Spinach nitrite reductase, J. Biol. Chem.
252 (1977) 896–909.
[39] W.G. Zumft, Ferredoxin: nitrite oxidoreductase from Chlorella
puriﬁcation and properties, Biochim. Biophys. Acta 1040 (1990)
237–244.