J. Biol. Macromol., 13(3), 78-85, 2013

Covalent Chromatography for Chymotrypsin-like Proteases Using a Diphenyl
1-Amino-2-phenylethylphosphonate Derivative
Shin Onoa,*, Junya Muraia, Seigo Furutaa, Kazuya Doikea, Fumie Manzakia, Toshiaki Yoshimuraa,
Hirofumi Kurodab, Masahito Umezakic and Hiroshi OyamadaGraduate School of Science and Engineering, University of Toyama, Toyama 930-8555, JapanbDepartment of Applied Chemistry and Chemical Engineering, Toyama National College of Technology,

Toyama 939-8630, Japan
cInstitute of Natural Medicine, University of Toyama, Toyama 930-0194, JapandFaculty of Science and Engineering, Setsunan University, Osaka 572-8508, Japan

Recieved October 15, 2013; Accepted November 16, 2013

To establish a covalent chromatography system for purification of naturally occurringchymotrypsin-like serine proteases, a diphenyl 1-amino-2-phenylethylphosphonate derivative
bearing Gly-Gly-Gly as a spacer was immobilized on the Sepharose 4FF gel. In this system, bovine
chymotrypsin was selectively bound to the phosphonate immobilized on the gel and then released by
the action of 2-pyridinealdoxime methiodide as the reactivated enzyme. Based on the study results
for selective binding and reactivation conditions, chymotrypsin-like proteases from pancreatin (hogpancreas) were rapidly and highly purified within three hours.

Keywords
chymotrypsin-like serine proteases, covalent chromatography, diphenyl 1-aminoalkylphosphonate,
immobilization, 2-pyridinealdoxime

Introduction

increased stability in aqueous solutions when

Diphenyl -aminoalkylphosphonates are known tocompared to organophosphorous compounds, such

irreversibly and specifically inactivate serine

as diisopropylphosphofluoridate. Powers et al.

proteases by forming a covalent bond as shown insucceeded in the synthesis of phosphonate

Fig. 1 [1]. The phosphonates have been applied to

conjugates with biotin or fluorescent compounds

severaltheiruseful for the investigation of localization ofirreversible inactivation reactions. The utility of

intracellular serine proteases [2-5]. We also

the phosphonates stems from their relatively

reported the selective removal of chymotrypsin*Corresponding author
Tel: +81-76-445-6845, Fax: +81-76-445-6697,
E-mail: shinono@eng.u-toyama.ac.jp

Abbreviations
2PAM, 2-pyridinealdoxime methiodide; Csin,
chymotrypsin; Tsin, trypsin;

biologicalsystems

throughS. Ono et al.

79

Figure 1. Inactivation reaction of the Phe type phosphonate against chymotrypsin and its reactivation
by 2PAM.

(Csin) by using diphenyl 1-amino-2-phenylethylphosphonate (Phe type phosphonate) [6, 7].
The phosphonates can be also used forthe purification of enzymes by making use of theirreversible character. Ashani et al. immobilized
2-aminoethyl p-nitrophenyl methylphosphonate on

Figure 2: Chemical structure of the the Phetype phosphonate bearing Gly3 spacer (G3P).

Sepharose 4B (GE Healthcare, Little Chalfont,
England) and purified acetylcholinesterase by

was synthesized (Fig. 2) and immobilized onusing 2-pyridinealdoxime methiodide (2PAM) as

Sepharose 4FF gel (GE Healthcare). The obtained

an activator that broke and replaced the covalent

gel was designated as G3P-gel. Previously, the Phebond between the phosphonate and enzyme [9].

type phosphonate was immobilized on Sepharose

Although purification was satisfactorily pursued,

4B gel using 6-aminohexanoic acid as a spacer for

the

of

the selective removal of Csin [6]. However, Csin

acetylcholin esterase was relatively long, e.g.,

captured by the gel could not be reactivated64−112 h with a 48−57% yield. The authors

effectively by the addition of 2PAM (data notsuggested the possibility of using the covalent

shown). The poor recovery of Csin might resultchromatography system for purifying other serine

fromproteases. However, to our knowledge, no6-aminohexanoyl group. Hence, we chose theeffective purification system for serine proteases

Gly-Gly-Gly spacer which is more hydrophilicthat uses the phosphonates has been reported.

than 6-aminohexanoic acid, in order to enhancetimerequiredforthereactivation

In this study, to establish a covalentchromatography system for serine proteases thatthehydrophobic

characteroftheaccess of 2PAM to the phosphorous atom at theactive site.

uses phosphonates, a Phe type phosphonate

Using a mixture of bovine Csin andderivative (G3P) bearing Gly-Gly-Gly as a spacer

trypsin (Tsin), the selective binding ability of80

Covalent Chromatography for Chymotrypsin-like Proteases
G3P-gel against Csin and the reactivation behavior

Synthesis of G3P

of Csin bound to the gel by 2PAM were examined.

Thr

Phe

type

phosphonate

wasFinally, the availability of the G3P-gel was

synthesized according to the method proposed byevaluated using a crude enzyme mixture from

Oleksyszyn

pancreatin that originated from the hog pancreas asderivative of the Phe type phosphonate wasa source of naturally occurring Csin-like serine

synthesized via the -aminoalkylation of triphenyl

proteases. The isolated product was characterized

phosphitebyphenylacetaldehyde in acetic acid. The obtained

enzymatic

activity,

sulfate-polyacrylamide

sodiumgeldodecylelectrophoresis(SDS-PAGE), and LC-MS/MS analysis.

[9].

An

with

phosphonate

wasN-benzyloxycarbonyl

benzylcoupledcarbamate

with

and

N-blocked

tripeptide Boc-Gly-Gly-Gly and the deprotectionof the Boc group gave the desired spacer-binding

Materials and methods

derivative G3P. The final product was purified byMaterials

silica gel chromatography and identified by
Pancreatin was purchased from Wako

1H-NMR and ESI-TOFMS.

Pure Chemicals Industries Ltd. (Osaka, Japan).
N-Benzoyl-L-arginine-p-nitroanilide(BAPNA)

Immobilization of G3P on Sepharose 4FF gel

N-benzoyl-L-tyrosine-p-nitroanilide

G3P (1 mg) was immobilized on the(BTPNA) were purchased from Peptide InstituteN-hydroxysuccinimide (NHS)-activated SepharoseN-Tosyl-L-lysine

4FF gel (0.7 ml) in 0.1 M NaHCO3 coupling

chloromethyl ketone (TLCK)-treated Csin andbuffer at a pH of 8.0 by rotation. To block the

N-tosyl-L-phenylalanine

ketoneremaining active NHS-groups, the gel was(TPCK)-treated Tsin were purchased from Sigma

incubated in 0.1 M Tris-HCl buffer for 3 h,

Chemicals (St. Louis, MO, USA).

followed by washing with 10 ml of the same buffer.

and

Inc.

(Osaka,concentrations

Japan).

werechloromethyl

determined

Proteinwitha

The resultant G3P-gel was stored in 50 mM

bicinchoninic acid (BCA) protein assay kit

ammonium acetate buffer (pH 4.5) at 4 °C. The(Thermo Fisher Scientific, MA, USA). Other

NHS-activated Sepharose 4FF gel was similarly

chemicals were reagent grade.

treated with 0.1 M Tris-HCl buffer (pH 8.5) alone,

Enzymatic activity was monitored on a
Hitachi U-1800 spectrophotometer (Tokyo, Japan)
equipped

withathermostated

Molecularionmassesphosphonate as the reference.

holder.

The amount of the Phe type phosphonate

synthesized

introduced on the gel was determined by thecompounds were determined with electrospray

quantitative analysis of the phenol released fromionization

the phosphonate during alkaline hydrolysis, using

time-of-flight

of

the

mass

cell

to prepare a control gel without Phe typespectrometer(ESI-TOFMS) on a Hitachi NanoFrontier LC-MS

reversed phase HPLC.

system (Tokyo, Japan). Reversed-phase HPLC
analysis was performed using a Shimadzu HPLC

Assay of enzymatic activity

system consisting of two LC-10ADvp pumps andan SPD-10Avp UV-Vis detector (Kyoto, Japan).

Csin and Tsin activities were assayed
using

BTPNA andBAPNA assubstrates,

S. Ono et al.

81

respectively. For Csin activity, a dimethylsulfoxide

enzymatic activities.

solution (100 l) of BTPNA was mixed with 880
l of 40 mM Tris-HCl (pH 7.8) containing 16 mM

Purification of Csin-like proteases from pancreatin

CaCl2 (Tris-buffer A) in a cuvette, and the mixture

For the preparation of a crude extract

was incubated for 5 min at 37 °C. An aliquot (20from pancreatin, 750 mg of pancreatin wasl) of the sample solutions to be assayed wassuspended in 15 ml of Tris-buffer A for 30 min at

added to the mixture, giving a 1 ml total volume of

4 °C. After centrifugation at 21,400 g and 4 °C for

the reaction mixtures. For Tsin activity, 10 l of

15 min, the obtained supernatant was used as thethe sample solutions and 890 l of Tris-buffer A

crude extract from pancreatin. The crude extract

were used. The final concentrations of BTPNA(10 ml) was loaded onto 0.3 ml of G3P-gel in aand BAPNA were 0.2 mM and 0.7 mM,

column (0.6 × 5 cm). The flow was circulated

respectively. The production of p-nitroaniline wasuntil no more decrease in Csin activity in theevaluated by monitoring the absorbance at 405 nm

extract was observed. The gel was then washedfor 5 min. The enzyme unit (U) was expressedwith 6 ml of Tris-buffer B. The bound components

with the hydrolysis unit calculated using thewere eluted by circulating 3 ml of the reactivation

following equation: U = A405/min (p-nitroaniline

solution for 30 min at 25 °C. This elution was

molar extinction coefficient = 9920 [10]).

repeated again, and each effluent was subjected togel-filtration chromatography using a HiTrap

Inactivation and reactivation procedure for Csin

Desalting column (1.6 × 2.5 cm, GE Healthcare) tofrom a model enzyme mixture by G3P-gel (batch)

remove 2PAM. The fractions exhibiting Csin

To

the

model

mixture

(0.25

ml)

activity were collected and stored at 4 °C as thecontaining bovine Csin (1 mg/ml) and Tsin (1finally purified product. The crude extract was alsomg/ml) in Tris-buffer A, G3P-gel (50 l) was

treated with 0.3 ml of the gel in the batch method

added in the plastic column and the suspension

described above to purify Csin-like proteases.

was gently mixed by rotation at 25 °C for 15 min(contact step). This procedure was repeated threetimes.

Washing with 0.25 ml of 40 mM Tris-HCl

Electrophoresis
SDS-PAGE

wasperformed

under(pH 7.8) containing 0.4 M NaCl (Tris-buffer B)

non-reducing conditions, as suggested by Laemmli

was repeated five times. After discharge of the[11]. The gel was stained with the Coomassie

solution, the remaining Csin and Tsin activities inbrilliant blue (CBB) reagent (Atto Co., Tokyo,

the effluent were measured. To the remaining gel,Japan). For fluorescence detection, the samples

0.25 ml of the reactivation solution (200 mM

were incubated with the fluorescein-labeled Phe

2PAM in Tris-buffer A) was added and thetype phosphonate [3] in Tris-buffer A for 20 min atsuspension was gently mixed for 20 min at 25 °C

25 °C before treatment with the SDS reagent.

to recover the components covalently attached to

After electrophoresis, detection was directly

the gel (reactivation step). The reactivation step

performed

was repeated three times, and the effluents wereblue-light-transilluminator (Invitrogen, Carlsbad,

collected individually to measure their residualUSA) with emissions at around 460 nm, and the

using

a

Safe

Imager2.0

82

Covalent Chromatography for Chymotrypsin-like Proteases
gel was then stained with the CBB reagent.

Mass spectrometry
The two protein bands (29 and 28 kDa) weresliced into small pieces (~1 mm) and placed into
1.5 ml siliconized tubes individually. Afterwashing with 25 mM ammonium bicarbonate
containing 50% acetonitrile to remove CBB, thegel pieces were dehydrated and dried in a speed
vacuum. To each tube, 100 l of 25 mM
ammoniumbicarbonate

containing

10mM

dithiothreitol (DTT) was added and incubated at

Figure 3: Selective binding of bovine Csin
(black) and Tsin (grey) in a model mixture to
G3P-gel and reactivation of the bound Csin by
2PAM.

56 °C for 1 h. After washing with the ammonium
bicarbonate buffer (100 l), 100 l of 55 mM

from a model mixture

iodoacetic acid in the same buffer was added.

The

optimized
of

Csin

using

conditionsfor

G3P-gelwereAfter reaction at 25 °C for 45 min, the gel pieces

purificationin the tubes were washed with the ammonium

determined in a model mixture system (Fig. 3). At

bicarbonate buffer, followed by dehydration. The

first, the effect of the contact time (0–20 min) on

obtained gel pieces were subjected to tryptic

the inactivation of Csin by the gel was examined.

digestion at 37 °C overnight. The degraded

Since no change in the residual activities between

fragments were extracted with 0.1% TFA and each

15 and 20 min of contact time was observed (dataextracted solution was desalted by ZipTipC18

not shown), 15 min of contact time was chosen for(Millipore) before LC-MS/MS analysis. The

further experiments. After the contact procedure

prepared samples were analyzed in positive mode

was repeated four times, Csin was selectively

and MS/MS data was collected. MS/MS ion searchbound to the gel (contact step). Secondly,

was performed by MASCOT.

non-specifically bound Csin and Tsin were almost
removed by washing (3 times). From these results,

Results

the Csin binding capability was estimated to be 21

Characterization of G3P-gel

U/ml of the gel by using the hydrolysis unit

The amount of the Phe type phosphonate

against BTPNA. In addtion, the repeated 2PAM

immobilized on G3P-gel was estimated to be 1.2

treatment (3 times) led to the 49% reactivation ofmol/ml of gel based on the amount of the phenol

Csin. On the other hand, less than 1% of Tsinreleased from the gel. The gel was stored in 50

activity was detected in the reactivated product.

mM ammonium acetate at 4 °C. Even after storing

These results indicate that Csin can be selectively

for two months under these conditions, the gel

purified using G3P-gel in the batch method.

showed effective binding ability against Csin.
Purification of Csin-like serine proteases from
Selective inactivation and reactivation of Csin

pancreatin

S. Ono et al.

83

Table 1. Purification of Csin-like serine proteases
A

from pancreatin using G3P-gel.

29 kDa component: chymotrypsin-C-like [Sus scrofa]
1 MLGITVLAAL LAYASSCGVP SFPPNLSARV VGGENAIPHS WPWQISLQYL
51 SGDTWKHTCG GTLITSTHVL TAAHCISNSR TYRVALGKNN LEVEDEEGSL
101 VVGVDSIFVH EKWNSLLIRN DIALIKLAEP VELSDTIQVS CLPEEGSLLP
151 QDYPCYVTGW GRLWTNGPIA AELQQGLQPV VDHATCSQRD WWGSTVRDTM
201 VCAGGDGVIS ACNGDSGGPL NCQAENGSWE VRGIVSFGSG LGCNTYKKPT251 VFTRVSAYID WIDQKIQL
B
28 kDa component: chymotrypsinogen B2-like isoform 2 [Sus scrofa]
1 MAFLWILSCF ALVGTAFSCG VPAIPPVLSG LSRIVNGENA VPGSWPWQVS
51 LQDGTGFHFC GGSLISEDWV VTAAHCGVTT SDVVVAGEYD QASDAEDIQVaCsin-like enzymatic activity was assayed using
BTPNA as a substrate. One unit of enzymatic
activity was defined as the hydrolysis of 1 mol of
BTPNA per min, using 9920 M-1cm-1 as theextinction coefficient of p-nitroaniline at 405 nm.

101 LKIAKVFKNP NFSLLTVRND ITLLKLATPA RFSRTVSAVC LPSASDDFPA
151 GTLCATTGWG KTKYTALKTP DKLQQAALPI VSSTVCKSYW GSKVTDVMIC
201 AGASGVSSCM GDSGGPLVCQ KNGAWTLVGI VSWGSSTCST TTPAVYARVT251 ALIPWVQQIL ANN

Figure 5: Identification of the 29 (A) and 28 kDa(B) components in the finally purified product by
LC-MS/MS. The results of MS/MS analysis forthe 29 and 28 kDa components matched thesequences of chymotrypsin-C-like protease(accession
no.
XP003127677)
andchymotrypsinogen B2-like isoform 2 (accession
no. XP003355797) from Sus scrofa, respectively.
The peptide sequences detected by MS areunderlined.

implying that the component was purified 17-folds
from the crude extract in the batch method.
Moreover, in case of the flow method, the final
Figure 4: Non-reducing SDS-PAGE analysisof the final product (3 g, B and D) and thecrude extract (5 g, C and E) with standard
protein molecular weight markers (A). Lanes(A-C) were stained with CBB after lanes (Dand E) were fluorescently detected.

product was estimated to be 1.4 U/mg, implyingthat the Csin component was purified 30-fold. On
the other hand, the Tsin-like enzymatic activity(BAPNA hydrolyzing activity) was undetectable.
The total time for the purification was about 3
hours.
The

To demonstrate the effectiveness of

SDS-PAGE

analysisunderG3P-gel on the purification of Csin-like serine

non-reducing conditions showed that the final

proteases,

product

the

crude

enzymeextract

fromcontained

threecomponents

withpancreatin was treated with the gel by twomolecular masses of 29, 28, and 23 kDa (lane B in

methods (batch and flow). As shown in Table 1,

Fig. 4). These three components were also

the specific activity of the Csin-like enzyme

detected by the fluorescein-labeled Phe typecomponent (BTPNA hydrolyzing activity) by thephosphonate,batch method was estimated to be 1.3 U/mg,

Csin-like serine proteases in a manner similar to

which

could

selectively

label

84

Covalent Chromatography for Chymotrypsin-like Proteases
that reported by Abuelyaman et al. [3], as shown inin fish. This is due to the limited affinity ligands

Fig. 4 (lane D). These findings indicate that theforCsin-like

proteases.

In

our

components corresponding to the three bands inchromatographysystem,slightamountstheCsin-likeCsin-like proteases can be highly purified in one

proteases. In particular, two components (29 andstep. Even if the target proteases present in low

28 kDa) seemed identical to the ones observed forconcentration, G3P-gel can capture them by

the crude extract (lane E), showing that these twocirculating the sample solutions (flow method). In

components would be Csin-like proteases fromaddition, other dominant components can be easily

pancreatin. It has been known that there areremoved by washing without loss of the target

several serine proteases in hog pancreas that canproteases because Csin-like proteases covalently

hydrolyze the Phe-X bond, e.g., Csin-A, -B, -C,

fixed on the gel.

final

product

originatefromcovalentofand elastase 2 [12]. In addition, the 23-kDa

The system presented here can be also

component may be a degradation product from theapplicable to the purification of other serine

29-kDa component because by comparison of theproteases.

bands observed for the crude extract (lanes D and1-aminoalkylphosphonatesE), the fluorescence intensity of the 29-kDa bandvarious amino acids can be synthesized [1],was reduced while the 23-kDa band was newly

replacement of the Phe type phosphonate moiety

apparent. The LC-MS/MS analysis (Fig. 5) for the(P1 position) by other phosphonates will lead tocomponents of the final product suggested that thethe preparation of gels available for the separation

29- and 28-kDa components would be Csin-C andof

Csin-B, respectively. Therefore, all these results

specificities. Moreover, the elongation of amino

demonstrated that the naturally occurring Csin-likeacid residues to the P2 and P3 positions in theproteases can be rapidly and highly purified from

phosphonate derivatives should be effective inpancreatin by using G3P-gel.

improving

Sinceserineproteases

thediphenylcorresponding

withdesiredselectivity.

This

to

substrate

covalent

chromatography system using a well-designed
phosphonate derivative will enable the recoveries

Discussion
Wedemonstrated

that

the

covalent

chromatography system using a combination ofof highly purified serine proteases without
excessive time-consumption.

G3P-gel and 2PAM was useful for purifying
Csin-like serine proteases. Many Tsin-like serine

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