Cross-Linking of Protein Subunits by1,3,5-Triacryloyl-hexahydro-s-triazine

Gervydas Dienys,‡ Jolanta Sereikaite,*,§ Gintaras Gavenas,§,| Rimantas Kvederas,§ andVladas-Algirdas Bumelis§

Faculty of Chemistry, Vilnius University, Lithuania, and Institute of Biotechnology, Graiciujno 8,Vilnius 2028, Lithuania. Received March 18, 1998; Revised Manuscript Received July 2, 1998

Six difunctional and trifunctional derivatives of acrylamide were synthesized and investigated aspotential protein lysine residue cross-linking agents. 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT) wasconsidered the best. The rate constants for the reactions of TAT with model nucleophiles in watersolution at 25 °C were with the glycine anion amino group, 7.69 × 10-3 M-1 s-1; with the anionic formof the N-acetyl-L-cysteine thiol group, 5.54 M-1 s-1; and with the NR-acetyl-L-histidine imidazole ring,1.19 × 10-5 M-1 s-1 (at pH 9.0). The kinetics of modification of amino groups by TAT were studiedfor several proteins: R1-casein, bovine serum albumin, recombinant human growth hormone,recombinant human interferons-R2b, and -γ. The results indicate that if proteins are associated intooligomeric structures in water, their subunits are effectively cross-linked by TAT.

Activated ethylenes of the general formula CH2dCH-X (X ) CN, COOR, CONH2, COR, NO2, SO2R, etc.)can be used in the chemical modification of proteinnucleophilic functional groups. Acrylonitrile (CH2dCH-CN) is considered the principal representative of thegroup (1, 2). Systematic investigation of interactionsbetween acrylonitrile and amino acids and peptides ormodel compounds have previously been reported byFriedman and his collaborators (2-7), who demonstratedthat of the nucleophilic groups of proteins thiol groupsare modified most readily. Amino groups are less reac-tive by 2-3 orders of magnitude, and the imidazolegroups of histidine are still less reactive (8). Otherfunctional groups of proteins are not modified.

In addition, thiol groups (7, 9, 10) and amino groups(11) of proteins have been demonstrated to be successfullymodified by acrylamide (CH2dCH-CONH2), though thereaction is slower (approximately by a factor of 10) thanthat with acrylonitrile. As a result, â-carbamidoethylgroups (CH2CH2CONH2) are introduced into the proteinmolecule. These groups closely resemble glutamineresidues. Neither protein electric charge nor pI isaltered, nor is solubility decreased, a common result aftermodification by acrylonitrile or alkylating agents (11).Acrylamide therefore is seen as an agent for the “delicate”modification of proteins. Finally, there are also publica-tions on the modification of proteins by several otheractivated ethylenes: methylacrylate (2, 7), 4-vinylpyri-dine (2, 12), and alkylvinylsulphones (2, 13, 14).

A large variety of N-substituted acrylamides can besynthesized easily (15). Reactivity of their ethylenicbonds in nucleophile-driven addition reactions variesconsiderably depending on the nature of N-substituents(16). We therefore investigated several bifunctional and

trifunctional derivatives of acrylamide as potential cross-linking agents for protein lysine residues. 1,3,5-Triacryl-oyl-hexahydro-s-triazine (TAT)1 was found to be a verypromising agent because of its high activity and stabilityin water solutions.

MATERIALS AND METHODS

Chemicals. Human serum albumin from Calbiochem,bovine serum albumin, myoglobin, acrylamide, N,N′-diacryloyl-diaminomethane, N-acetyl-L-cysteine, dithio-threitol, and 2,4,6-trinitrobenzene sulfonic acid fromServa, R1-casein from Sigma, and NR-acetyl-L-histidinefrom Reachim (Russia) were used. Recombinant humaninterferon-R2b (IFN-R2b), recombinant human interfer-on-γ (IFN-γ), recombinant tumor necrosis factor R (TNFR),and recombinant human growth hormone (hGH) wereproducts of Biofa AB (Lithuania). Protein markers forsodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) from Sigma, Pharmacia, and Bio-Radwere used.

1H-NMR Analysis. 1H-NMR spectra were recordedon a Hitachi 22 spectrometer, operating at 90 MHz, usingtetramethylsilane as the internal standard.

Synthesis of Cross-Linking Agents. N,N′-Diacryl-oyl-diamines (Table 1, nos. 3-5). Acryloylchloride (8mL, 0.10 mol) was slowly added with stirring to a mixtureof diamine (0.05 mol), anhydrous sodium acetate (9 g,0.11 mol), chloroform (50 mL), and hydroquinone (0.05g) at 10 °C. The reaction mixture was stirred for 2 h atroom temperature and then was filtered. The filtrate wasleft overnight at 4 °C. A white precipitate was collectedand recrystallized from chloroform or hexane.

N,N′-Diacryloyl-1,2-diaminoethane. Yield, 5 g (60%);mp, 145 °C [lit. (17) mp, 144.5-145 °C]. 1H-NMR (CD3-

* Author to whom correspondence should be addressed.Phone: (370 2) 642514. Fax: (370 2) 642624. E-mail: Sereik@ibt.lt.

‡ Vilnius University.§ Institute of Biotechnology.| Present address: BIOK Ltd., P.O. Box 2546, Vilnius,

Lithuania.

1 Abbreviations: TAT, 1,3,5-triacryloyl-hexahydro-s-triazine;IFN-γ, recombinant human interferon-γ; IFN-R2b, recombinanthuman interferon-R2b; hGH, recombinant human growth hor-mone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; BSA, bovine serum albumin; TNFR, recombi-nant tumor necrosis factor R.

744 Bioconjugate Chem. 1998, 9, 744−748

10.1021/bc9800318 CCC: $15.00 © 1998 American Chemical SocietyPublished on Web 09/25/1998

SOCD3): δ 3.43 (m, 4H, CH2CH2), 5.67 (q), 6.76 (m, 6H,CHdCH2), 8.40 (br s, 2H, NH).

N,N′-Diacryloyl-1,3-diaminopropane. Yield, 7.9 g(87%); mp, 109-110 °C. 1H-NMR (CD3SOCD3): δ 1.88(m, 2H, CH2), 3.35 [m, 4H, (NCH2)2], 5.76 (q), 6.30 (q,6H, CHdCH2), 8.43 (br s, 2H, NH). Anal. Calcd forC9H14N2O2: C, 59.32; H, 7.74; N, 15.37. Found: C, 59.09;H, 7.85; N, 15.16.

N,N′-Diacryloyl-piperazine. Yield, 6.3 g (65%); mp,91-92 °C [lit. (18) mp, 98 °C]. 1H-NMR (CD3SOCD3): δ3.76 (s, 8H, piperazine ring), 5.91 (q), 6.31 (q), 7.04 (q,6H, CHdCH2).

1,3,5-Triacryloyl-hexahydro-s-triazine (TAT, Table1, no. 6). TAT was prepared as described by T. L.Gresham and T. R. Steadman (19) and recrystallizedfrom water. TAT polymerized when heated and nodefinite melting point could be identified. 1H-NMR(CDCl3): δ 5.39 (s, 6H, CH2), 5.80 (q), 6.32 (q), 6.78 (q,9H, CHdCH2).

Diacrylimide (Table 1, no. 7). Diacrylimide wasprepared as previously described (20). The product waspurified by column chromatography on silica gel usingethyl acetate/chloroform (1/1, v/v) as the eluent. Di-acrylimide was crystallized from water: mp, 179 °C [lit.(20) mp, 180 °C]. 1H-NMR (CDCl3): δ 5.82 (q), 6.42 (q),6.81 (q, 6H, CHdCH2).

Kinetic Measurements. The kinetics of reactionsshown in Scheme 1 were followed spectrophotometricallyin thermostated cell at 255 nm for unsaturated amidesno. 1-5 and 7 (Table 1) and at 260 nm for TAT (no. 6).The extinction coefficients of amides no. 1-5 and 7 at 255nm were 134, 1260, 1680, 1470, 8340, and 2530 M-1 cm-1,respectively, and that of TAT at 260 nm was 3120 M-1

cm-1. The optical density of the piperidine and glycine

at 255 and 260 nm was neglible. The extinction coef-ficient of piperidine at 255 nm was 1.46 M-1 cm-1. UV-absorption spectra of addition products were shiftedtoward the short-wave side with respect to the spectraof initial unsaturated amides. From 6 to 10 kinetic runswere carried out for each reaction. Molar concentrationof the product at the time t(x) was calculated by theequation

where A is the absorbance at the time t, Ao is the initialabsorbance and Ainf is the final absorbance, when thereaction is completed, a is the initial concentration ofN-substituted acrylamide.

The kinetics of the reactions shown in Scheme lB werestudied in alkaline solutions at several pH values andrate constants were calculated for anion NH2CH2COO-

as the nucleophile. The kinetics of alkaline hydrolysisof diacrylimide were also investigated spectrophotometri-cally at pH values between 10.05 and 13.6.

The kinetics of modification of protein amino groups(Scheme 3, Table 2) were followed by measuring theconcentration of free amino groups in aliquots of thereaction mixture every 30 min over a period of 3-4 h,using the 2,4,6-trinitrobenzene sulfonic acid method (21)as described by Jonusiene et al. (22). Three kinetic runswere carried out for each reaction. Second-order rateconstants were calculated from the reaction rate valuesobtained by the method of cubic smoothing splines (23)at the initial moment and after modification of 50% ofthe amino groups.

The kinetics of the reaction of TAT with N-acetyl-L-cysteine thiol groups were followed spectrophotometri-

Table 1. Rate Constants k (M-1 s-1) for Reactions of Potential Cross-Linking Agents with Model N-Nucleophiles inWater Solution at 25 °C

N-nucleophile

derivative of acrylamide structure

NH

k × 103 aNH2CH2COO-

k × 105 a

1 acrylamide CH2dCHCONH2 34.00 ( 3.5b 38.30 ( 2.2b

2 N,N-diacryloyl-diaminomethane CH2dCHCONHCH2NHCOCHdCH2 16.66 ( 1.43 32.54 ( 2.593 N,N-diacryloyl-1,2-diaminoethane CH2dCHCONHCH2CH2NHCOCHdCH2 5.06 ( 0.50 7.53 ( 0.584 N,N′-diacryloyl-1,3-diaminopropane CH2dCHCONH(CH2)3NHCOCHdCH2 2.79 ( 0.15 5.52 ( 0.545 N,N′-diacryloyl-piperazine

N

N

COCH

COCH

CH2

CH2

29.07 ( 2.37 63.10 ( 2.75

6 1,3,5-triacryloyl-hexahydro-s-triazine

N

NN COCHCHCO

COCH CH2

CH2CH2752.00 ( 46.67 769.00 ( 47.33

7 diacrylimide CH2dCHCONHCOCHdCH2 6123 ( 201 14590 ( 1300a Rate constants for one electrophilic group (CH2dCHCO-) are given. b Standard deviations are given.

Scheme 1

Scheme 2

x ) a(Ao - A/Ao - Ainf)

Cross-Linking of Protein Subunits Bioconjugate Chem., Vol. 9, No. 6, 1998 745

cally in a thermostated cell, at 275 nm. Six kinetic runswere carried out for each experiment. The second-orderrate constants for the N-acetyl-L-cysteine dianionic formwere calculated from initial rate values at 6.5, 10.5, and14.5 °C: 2.67 ( 0.08 M-l s-1, 3.26 ( 0.22 M-1 s-1 and3.69 ( 0.51 M-1 s-1, respectively. The rate constant at25 °C was calculated using an Arrhenius plot, 5.54 M-1

s-1.The kinetics of the reaction of TAT with the imidazole

ring of NR-acetyl-L-histidine at pH 9.0 was also followedspectrophotometrically in a thermostated cell at 275 nm,under pseudo-first-order conditions (150-fold excess ofNR-acetyl-L-histidine). Three kinetic runs were carriedout for each experiment. The second-order rate constantscalculated at 60, 70, and 80 °C were (2.32 ( 0.39) × 10-4

M-1 s-1, (3.98 ( 0.56) × 10-4 M-1 s-1, and (9.31 ( 0.33)× 10-4 M-1 s-1, respectively. The rate constant at 25 °Cwas calculated using an Arrhenius plot, 1.19 × 10-5 M-1

s-1.Cross-Linking of Oligomeric Proteins by TAT.

Protein solutions were prepared in 0.05 M borate buffer,pH 9.2, such that the initial concentration of aminogroups was between 0.06 and 0.59 mM. TAT was addedin 4.5-67-fold molar excess with respect to the concen-tration of amino groups in the protein solution. Thereaction mixture was incubated at 30 °C. Aliquots werewithdrawn over the course of the reaction, and thereaction was stopped by the addition of excess cysteineor dithiothreitol. Samples of the reaction mixture weredialyzed and analyzed by SDS-PAGE for identificationof cross-linked protein forms.

SDS-PAGE. SDS-PAGE was carried out on theMighty Small electrophoresis unit (Hoefer ScientificInstruments) according to the method of Laemmli (24).The acrylamide concentrations used in the gel were12.5% (for hGH, TNFR), 15% (for INF-γ, myoglobin), and7.5% (for human serum albumin). Proteins were stainedwith Coomassie Brilliant Blue R-250 and the gels werescanned at 633 nm using the Dual-Wavelength TLCscanner (CS 930, Shimadzu).

RESULTS AND DISCUSSION

Investigation of Cross-Linking Agents. Six di-functional and trifunctional derivatives of acrylamidewere synthesized. Their activity in nucleophilic additionreactions was evaluated by measuring the kinetics of the

reactions with low molecular weight model N-nucleo-philesspiperidine and the anion of glycine (Scheme 1).The rate constants measured are given in Table 1. Forcomparison, acrylamide is included together with itspolifunctional derivatives.

N,N′-Diacryloyl-diaminoalkanes (Table 1, nos. 2-4)were found to be less reactive than acrylamide. They areof little interest as cross-linking agents, however, becausetheir rate of modification is too low for practical purposes.The half-life of the most reactive protein amino groupsat 25 °C is over 6 h if a concentration of acrylamide of0.1 M is used (11).

Diacrylimide was the most active of the tested com-pounds (Table 1, no. 7). It is not, however, stable inalkaline solutions. It is hydrolyzed to form acrylamideand acrylate (25). Diacrylimide is a weak acid, and inalkaline solution it ionizes partly to form the anion (CH2dCHCO)2N:-; a process which needs to be taken intoaccount when the kinetics of its hydrolysis and nucleo-philic addition of amines are studied. We measured thediacrylimide ionization constant (pKa ) 10.9) using thespectrophotometric method (26) and investigated thekinetics of its alkaline hydrolysis. The kinetic data areconsistent with the mechanism including, as the slowstep, the attack of diacrylimide unionized molecule byan hydroxide anion (Scheme 2). The rate constant of thereaction described under Scheme 2 at 25 °C in watersolution was 4.35 M-1 s-1, a value similar to or slightlyhigher than those for the hydrolysis of saturated im-ides: diacetimide (k ) 0.88 M-1 s-1), succinimide (k )3.14 M-1 s-1) (25). At pH 9.2, the half-life of diacrylimideis approximatly 3 h, at pH 10, approximately 30 min.Diacrylimide may therefore be of interest, under condi-tions where temporary cross-linking is required.

We found that the best cross-linking agent from thecompounds listed in Table 1 was TAT. Its activity wasconveniently high and its stability in water was remark-able. No change was identifed after storing TAT inbuffer, pH 9.2, over the course of 1 week at roomtemperature. Its three active groups make it an espe-cially effective cross-linking agent.

In order to characterize the reactivity of TAT, thekinetics of its reactions with two other model nucleophilesN-acetyl-L-cysteine and NR-acetyl-L-histidine were in-vestigated. The rate constant of the anionic form ofN-acetyl-L-cysteine (a model of thiol groups of proteins)was 5.54 M-1 s-1 at 25 °C in water, a value whichexceeded, by nearly three orders of magnitude, the rateconstant of the glycine anion amino group (Table 1).Such an activity ratio is supported by the literature (4).The histidine residue imidazole ring is not as active anucleophile as the amino group. The rate constant of thereaction between NR-acetyl-L-histidine and TAT at 25 °Cand pH 9.0 was 1.19 × 10-5 M-1 s-1. Consequently, it isclear that, if there are no free thiol groups, TAT is quitea specific modifying agent for protein amino groups.

Modification and Cross-Linking of Proteins. Thefirst step in the interaction between TAT and a proteinmolecule is generally the addition of an amino group toone of the acryloyl groups (Scheme 3). If free thiol groups

Scheme 3a

a P represents a protein molecule.

Table 2. Rate Constantsa and Half-Lives of ProteinAmino Groups Modification by TAT at pH 9.2 and 30 °C

proteinc[TAT]o(mM)

k × 103 b

(M-1 s-1)kl × 103 c

(M-1 s-1)half-lifed

(h)

Rl-casein 2.75 8.58 ( 0.43e 6.66 ( 0.71e 3.19 ( 0.27e

BSA 3.24 9.15 ( 0.80 4.19 ( 0.18 3.21 ( 0.22hGH 3.69 7.36 ( 0.58 6.15 ( 0.58 2.73 ( 0.29IFN-R2b 8.13 9.36 ( 0.88 8.06 ( 0.38 0.88 ( 0.05IFN-γ 8.13 15.11 ( 3.49 8.13 ( 1.62 0.69 ( 0.15

a Rate constants for one electrophilic group (CH2dCHCO-) ofTAT are given. b Rate constants calculated at the initial time.c Rate constants calculated after modification of 50% of the aminogroups. d Time required for modification of 50% of the aminogroups. e Standard deviations are given.

746 Bioconjugate Chem., Vol. 9, No. 6, 1998 Dienys et al.

are present, though, their reaction precedes that of theamino groups. Intra- and intersubunit cross-linking ofamino groups then follows.

We therefore studied the kinetics of the TAT-inducedmodification of the amino groups of several proteins(Table 2). It was found that the rate constants decreasedduring the addition reaction. Solubility of TAT in wateris limited (approximately 40 mM at room temperature).Half of the amino groups are modified in less than 15min in the presence of such a concentration of TAT at 30°C and pH 9.2. We therefore used mixed solvents water-methanol and water-ethanol to promote the modificationrate in some cases (27).

Cross-linking investigations were carried out withseveral proteins, both monomeric and oligomeric forms.The course of cross-linking was followed by SDS-

electrophoresis of samples of reaction mixtures. Mostexperiments were performed at pH 9.2 and 30 °C usinga 4.5-14-fold excess of TAT compared with the concen-tration of amino groups present. At pH 8.0, though,using the maximum possible concentration of TAT(approx. 40 mM) and leaving the reaction mixtureovernight at 30 °C practically complete cross-linkingcould also be achieved.

At the protein concentrations used (0.5-2.0 mg/mL),no cross-linking was registered for proteins which weremonomeric in water (see, for example, Figure 1, bottom).But after modification by TAT, electrophoretic bands ofproteins were slightly displaced and became broader. Thechange was greatest for the proteins having the greaternumber of Lys residues (human serum albumin, myo-globin). Proteins, that are dimeric in water [for example,IFN-γ (28)], were transformed by TAT into covalentlybonded dimers, which were not separated during SDS-electrophoresis (Figure 1, top).

It is known that Zn2+ ions induce the dimerization ofhGH (29), an observation confirmed by our measure-ments of the action of TAT. In the presence of Zn2+,cross-linked dimers were formed (Figure 1, middle), butno dimers were detected in the absence of Zn2+ (Figure1, bottom). The kinetics of hGH cross-linking are shownin Figure 2. If Zn2+ ions were added after amino groupshad been already partly modified by TAT (Figure 2,experiment B), formation of cross-linked dimers occurredmore rapidly, where Zn2+ and TAT were added together(Figure 2, experiment A). Apparently, the cross-linkingstep is faster than the addition of amino groups to TAT.If experiments were continued for long enough (24 h),the yield of dimer in both cases approached 100%.

TNFR is known as a trimeric protein in water (30).The time course of its cross-linking by TAT was alsoinvestigated (Figure 3). Polymerization was evident after30 min, when the dimeric form was clear, its presencewas maximal after 90 min, but at longer time intervalsthe trimeric form developed to become predominant,though two bands of molecular weight higher than thedimeric form were identifiable. Most likely, both repre-

Figure 1. (Top) SDS-PAGE (15%) of IFN-γ samples. Lane 1,protein markers; lane 2, unmodified IFN-γ; lanes 3-5, samplestaken at 15, 30, and 60 min. Concentration of IFN-γ, 0.16 mg/mL; initial concentration of amino groups, 0.18 mM; concentra-tion of TAT, 1.0 mM. (Bottom) SDS-PAGE (12.5%) of hGHsamples modified in the absence of Zn2+. Lane 1, unmodifiedhGH; lanes 2 and 3, samples taken at 60 and 120 min; lane 4,protein markers. Concentration of hGH, 0.75 mg/mL; initialconcentration of amino groups, 0.39 mM; concentration of TAT,4.5 mM. (Middle) SDS-PAGE (12.5%) of hGH samples modifiedin the presence of Zn2+. Lane 1, unmodified hGH; lanes 2 and3, samples taken at 60 and 120 min; lane 4, protein markers.Concentrations of hGH, amino groups and of TAT were the sameas those in the bottom panel. Molar ratio of Zn2+ to hGH in thereaction mixture was 2:1.

Figure 2. Kinetics of hGH cross-linking by TAT (0.05 M boratebuffer, pH 9.2, 30 °C; initial concentrations: hGH, 0.3 mg/mL,14 µM; TAT, 1.8 mM). Experiment A (4), Zn2+ (28 µM) wasadded together with TAT; experiment B (O), Zn2+ (28 µM) wasadded after 25% of amino groups were modified by TAT.

Cross-Linking of Protein Subunits Bioconjugate Chem., Vol. 9, No. 6, 1998 747

sent cross-linked trimers (31). A very weak band of amolecular weight corresponding to that of a hexamerappeared also at long time intervals. X-ray studiesindicate the assembly of two trimers of TNFR to make ahexameric unit in the crystal form (32). Dimerization oftrimers may also occur to some extent in solution.

Cross-linking of protein amino groups by TAT is a verysimple and rapid way to establish whether proteinoligomers are formed in solution. The high stability ofTAT in water promotes its utility. It would be verydifficult to conduct experiments similar to those shownin Figures 2 and 3 with any unstable cross-linking agent,for example, dimethyl suberimidate.

ACKNOWLEDGMENT

We thank Biofa AB for the gift of hGH, INF-R2b, INF-γ, and TNFR.

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BC9800318

Figure 3. SDS-PAGE (12.5%) of TNFR samples. Lane 1,unmodified TNFR; lane 2-12, samples taken at 15, 30, 45, 60,90, 120, 180, 240, 480 min, 24, and 48 h; lane 13, proteinmarkers. Concentration of TNFR, 0.15 mg/mL; initial concentra-tion of amino groups, 0.06 mM; concentration of TAT, 4 mM.

748 Bioconjugate Chem., Vol. 9, No. 6, 1998 Dienys et al.