J. Biol. Macromol., 13(1), 13-20 (2013)

ArticleSearch for pterin-binding protein from Euglena

Junko Takeda(1, *), Minori Nakashima(1), Hiroshi Ueno,(1) Tomoko Mori(2).
Mineo Iseki(3) and Masakatsu Watanabe(4)(1) Lab. of Appl. Microbiol. & Biochem., Nara Women’s Univ., Nara 630-8506, Japan(2) National Institute for Basic Biology NIBB Core Research Facilities Functional Genomics
Facility, Okazaki 444-8585, Japan(3) Dept. Pharmacol. , Toho Univ., Funabashi 274-8510, Japan(4) Grad. Sch. for Creation of New Photonics Indust., Hamamatsu 431-1202, Japan

Received February 22, 2013; Accepted March 19, 2013

BIAcore analysis with a biopterin (BP)-bound sensor chip could be a powerfultool for the search of pterin-binding proteins. Although preparation of the sensor chip iscrucial, a conventional amine coupling procedure is not applicable for the ligand such as
BP that has no primary amino group.

In this study, we describe the method to introduce

the primary amino group onto BP molecule efficiently.

Key words: Biopterin binding protein, biopterin sensor chip for BIAcore, organic synthesis ofprimary anino group to biopterin.

Introduction
Euglena gracilis, a unicellularfor over a century because it shows various
photomovement-responses such as phototaxis,

photosynthetic flagellate, has served as aphotokinesis and photophobic responses (1,model system for studying light-perception2). However, the responsible photo-sensor

and signal transduction in photo-responses

molecules except photoactivated adenylyl___________________________________*To whom correspondence should be
addressed,
telephone:+81-075-702-3417,
E-mail:jtakeda-chappy3@ares.eonet.ne.jpcyclase (PAC)(3) for the step-up photophobic
response have remained unidentified until
recently.

14Search for pterin-binding protein

In 2002, Iseki et al. isolated PAC

BP-binding protein from paraflagellar bodyfrom paraflagellar body (PFB) of Euglena.

(PFB)-rich fraction of Euglena. For simple

PAC is a heterotetramer composed of two

and rapid detection of BP-binding activity,

flavin adenine dinucleotide (FAD) binding

we considered the application of BIAcore

subunits of alpha and beta chains in twosystem that utilizes the surface plasmon

sensors of blue-light-using-FAD (BLUF)

resonance.

domains.

The RNA interference (RNAi)

primary amino group are immobilized onto

analysis indicates that PAC functions as aBIAcore sensor chips via amine coupling

receptor

forthestep-up

photophobic

method, whereas those ligands without

responseandnotforstep-down

primary amino group are immobilized via

photophobic response. The results suggest

their hydroxyl groups. With many attempts,

a presence of a different photoreceptor for thethe method described in BIA direction (7) has

step-down photophobic response.

Thenot been successful in producing a BP sensor

action spectrum for the step-up photophobic

chip. In this study, we introduce a method

responses

flavinwhere the primary amino group attaches toabsorption spectrum with two characteristic

one of the hydroxyl groups of the propyl-side

peaks at 350 nm and 450 nm in the equal

chainheight (4), while that for the step-down

conventional amine coupling method.

corresponded

theto

theofGenerally,

BP inligands

order tohaving

apply thephotophobic response showed two peaks
with unequal heights; the peak at 350 nm isMaterials and Methods

higher than that at 450 nm. This indicates

The reagents used were biopterin (BP),the presence of photoreceptor molecules

2,2’-ethylendioxy bis-(ethylamine (diamine)other than PAC, the molecules characterized(Sigma-Aldrich, Tokyo, Japan), disuccimidylby having an absorption maximum aroundcarbonate(DSC),

350 nm that was added to the flavin spectrum.

(DMAP),

andOne of such substances may be an oxidized(DMSO) (Wako Pure Chemical Industries

biopterin (BP)existence ofLtd, Osaka, Japan). These reagents as well

BP-binding protein in Euglena was suggested

as their stock solutions were stored in a(5,6). However, no method is available indesiccator before use.

sincethedimethylaminopyridine

drieddimethylsulfoxide

detecting the BP-binding activity with simple
and rapid way, which has prevented theHydrophilic

identification of BP-binding protein.

chromatography

We have attempted to isolate theinteraction

liquidBP and the reaction products in DMSOTakeda et al.

15

Figure 1 The reaction scheme for the introduction of primary amino group to BP.
The product, BP-O-C(=O)-NH-spacer-NH2 (MW 414.4), is indicated by the solid linebox. The compounds confirmed by MS spectrum are marked with yellow.
werefractionatedinteractionbyliquidthehydrophilic(PCHILIC) (Shiseido Co. Ltd Tokyo, Japan)

chromatography

under the following conditions: The column

Figure 2 HPLC Elution Pattern of 70% CH3CN for reaction
mixture before and after the reaction. Before: BP (a), DSC
and DMAP in DMSO were added to (a) at 2.5 days or more
(after the first step reaction completed)(b), and diamine wasadded to (b) at 30 min (second step reaction completed) (c),respectively. The vertical and horizontal axes show theabsorbance at 360 nm and time. Scales show 5 min. intervals

16Search for pterin-binding protein

size and eluents are 4.6 mm id x 250 mm,and 10 mmol L-1 HCOONH4 in 70, 80, orResults and Discussion

90% CH3CN, respectively.

Introduction of the primary amino group to

The flow rate,temperature, and absorption wavelength are
set at 1.0 mL min-1, 28 oC, and 360 nm,

one of the hydroxyl group of BP
The coupling reaction of a primaryamino group with an activated hydroxyl is arespectively.

urethane forming reaction (8). The reaction
scheme is illustrated in Figure 1.

MS and MS/MS analysis

TheMS and MS/MS analyses were

products confirmed to have the oxidized BP

performed using a Q-TOF mass spectrometerby the mass spectrometry （MS） are marked(Waters Co. Milford, USA).

The sample

with yellow, and the amino group attached

was diluted in two-fold with 0.1% HCOOH

BP, (NH2-spacer-(O)-BP), is indicated by aand applied to the MS spectrometer with asolid line box.

syringe pump.

attached BP has MW 411.4 and the molar

The primary amino groupratio of BP to diamine is 1:1. The reaction

Figure 3 MS/MS spectrum of m/z 412 which picked up from MS
spectrum of peak 1.
Fragment peaks of MS/MS for BP are indicated in yellow. Thefragments in red are derived from the partial structure –O-CH2
CH2-O-CH2CH2 NH2 in red font of inserted figure. Vertical andhorizontal axis show the relative abundance and m/z, respectively.

Takeda et al.

17In themaximum around 350-360 nm at neutral or

first step, one of the hydroxyl groups of BP

alkaline pH. The polarity of the reaction

was activated with both addition of DSC andproducts was expected to be higher than that

DMAP to form a succinimidyl-carbonyl-BP,

of BP as the primary amino group wasan activated BP complex.

incorporated.

BP was eluted at 6.3, 10.5

step, the activated BP complex couples toandmin

when

one of the primary amino groups of diamine

concentrationof70,

to form NH2-spacer-(O)-BP.

respectively, was used as an elution solvent.

is supposed to proceed in two steps.

In the second

12.0

CH3CN
80,

andwith90%,

The activation of hydroxyl group

Figure 2 shows the HPLC elution patterns

requires DSC and the reaction must bewith 70% CH3CN for (a) BP (b) the reaction

carried out in organic solvent since DSC is

mixture of BP, DSC and DMAP after the firsteasily hydrolyzed by water. We chose dry

step reaction reached at the maximum andDMSO as a solvent since BP was mostremained, and (c) the reaction mixture of (b)soluble in DMSO among the organic solvents

after the second step reaction.

examined.

In this reaction, 1) one of theindicates that BP is eluted at 6 min, Figure 2b

two hydroxyl groups on BP must be activated,shows the appearance of several inseparableand 2) the activated BP complex can easily

peaks as a cluster between 2.5 and 4.0

couple to one of the amino groups of diamine.

minutes, and in Figure 2c the cluster

The BP, DSC and DMAP were mixed withdisappeared and several peaks appeared.

their molar ratio of 1:4:1, respectively, andThe elution profiles suggest that the cluster isthenprobably the intermediate product having

anexcessamountofdiamine,

approximately 25 times as much as theMW

amount of the BP, was added.

succinimidyl-carbonyl-BP,

After the359.2,

whichanFigure 2aisactivatedreaction was completed, no byproducts wascomplex of BP, as shown in Figure 1. The

detected by the MS analysis for the reaction

first step reaction proceeded slowly, reachedmixture having the molar ratios of BP toat the maximum after 2.5 days, and remained

diamine as 1:2 and 2:1.

for more than three months in a desiccator

We have attempted to separate andwithout light.

During this reaction, theidentify the reaction products by HPLC using

amount of BP declined while that ofa PC HILIC column (Shiseido, Japan). The

inseparable cluster increased with time.

eluate was monitored at 360 nm as theBefore initiating the second step reaction bycompounds

pterinadding an excess amount of diamine, a small

absorption

amount of water was added to the reaction

structurehaving the oxidized
thatexhibitsthe18Search for pterin-binding protein

The peaks are named in descending order inheight as peak 1 at 18.5 min, peak 2 at 8 min
and peak 3 at 21 min.
The products at the BP peak as
well as the peak 1, peak 2 and peak 3 werecollected and analyzed by mass spectrometry(MS) to determine their structures. The MS
analysis indicated that the mass of the BP
peak was 238.0742, which agrees with thetheoretical mass of BP, 237.2. A largest peakat 412.1606 agrees with the mass for theexpected

BP-(O)-spacer-NH2(411.4)

obtained from peak 1-eluent and other peaks
are negligible (less than 10% of themaximum intensity). The peak at 412. wasgathered and further analyzed by MS/MS andFigure 4 Effects of pH on the second
step reaction.
The activated BP
complex in DMSO was added todiamine solution at pH8 (a) or pH10 (b).
After incubation at RT for 30 min, thereaction mixture was analyzed byHPLC.

88.072) may be originated from the partial

mixture to prevent further activation of BP.

structure as indicated with red fonts in theWhile the activation was inhibited by theFigure 3 insert.

presence of a trace amount of water, theoriginated fragments were detected from thereaction

largest peak at 432.0948 obtained from peak

product

immediately.

was

not

hydrolyzed

Only 10% of the reaction

shown in Figure 3, in which BP and BP
fragments are marked with yellow.

Thepeaks marked with red (106.0843, and2-eluent.

Neither BP nor BP

From peak 3-eluent the largest

product was hydrolyzed when 50% volume

peak at 412.1838 may be the expected BP-of water was added to the reaction mixture(O)-spacer-NH2

and kept overnight in the ice box.

products may be produced by the oxygenThesecond step reaction proceeded rapidly andwas completed within 30 minutes.

(411.4).

Thesetwoattach either at C9 or C10.
The MS and MS/MS analysisAsshown in Figure 2c, the cluster decreased

confirmedBP-(O)-spacer-NH2largely and three distinct peaks appeared.

synthesized as the peak 1 and peak 3 by ourwasdescribed method. We obtained a stableTakeda et al.

product, succinimidylcarbonyl-BP (activated19

grant # 18077003 to M. W.

BP), in the first step reaction in an organic
solvent

thoughtheconsiderably slowly.

reactionproceeded

Coupling of theactivated BP to the primary amino group inReferences(1) Lebert M. (2001) Phototaxis of Euglena

the second step reaction was expected togracilis- flavins

proceed rapidly at high pH. We compared

Comprehensive series in phorosciences

the reaction efficiency at pH 8 and pH 10(Hader D-P and Lebert M eds.) Vol. 1(Fig 4). There were no significant changes

Photomovement, pp 297-341, Elsevier.

in the peak of the activated complex whereas(2) Hader D-P, Ntefidou M. Iseki M. andthe product peaks were barely detected at pH

Watanabe M. (2005). In Light sensing in8 (a). At pH 10, on the other hand, the peakplants (Wada M., Shimazaki K. Iino.M.

of the activated complex decreased rapidly aseds)

the product peaks (peaks 1 and 3) grew, andISBN-4-431-24002-0

ppandpterins.

223-229,

InSpringer

the byproduct peak (peak 2 in Figure 2) was(3) Iseki M., Matsunaga S., Murakami A.,

undetectable (b). The area ratio of the BP

Ohno K., Shiga K., Yoshida K., Sugaiand product peaks (peaks 1 and 3) is 1:2.1,

M., Takahashi T., Hori T. and Watanabe

indicating a desirably high yield.

M.

Getting the BP derivative having(2002)

A

blue-light-activatedadenylcyclase mediated photoavoidance

the primary amino group can be purified by ainproper preparative column, the BP-sensor

1047-1051

Euglenagracillis.

Nature415

chip could be produced by the conventional(4) Matsunaga S., Hori T., Takahashi T.,

amine coupling method and used for isolating

Kubota M., Watanabe M., Okamoto K.,

the BP-binding proteins from Euglena.

Masuda K. and Sugai M. (1998)
Discovery of signaling effect of UV-B/CAcknowledgement

light in the extended UV-A/blue-type

Authors wished to thanks Drs.

action spectra for step-down and step-up

Satoshi Sakamoto in Tokyo Institute ofphotophobic response in the unicellularTechnology and Miki Akamatsu in Kyotoflagellate

University for giving advice in organic

Protoplasma 201 45-52

algaEuglenagracilis.

Authors indebted to Yumiko(5) Brodhun B. and Hader D-P. (1990)

Ueno for proofreading the manuscript. This

Photoreceptor proteins and pigments inwork was partly supported by the MEXT

the paraflagellar body of the flagellate

synthesis.

20

Search for pterin-binding protein

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