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Bioconjugate Chem. 1998, 9, 744−748
Cross-Linking of Protein Subunits by 1,3,5-Triacryloyl-hexahydro-s-triazine Gervydas Dienys,‡ Jolanta Sereikaite˘ ,*,§ Gintaras Gave˘ nas,§,| Rimantas Kvederas,§ and Vladas-Algirdas Bumelis§ Faculty of Chemistry, Vilnius University, Lithuania, and Institute of Biotechnology, Graicˇiu j no 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 as potential protein lysine residue cross-linking agents. 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT) was considered the best. The rate constants for the reactions of TAT with model nucleophiles in water solution at 25 °C were with the glycine anion amino group, 7.69 × 10-3 M-1 s-1; with the anionic form of 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 studied for 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 into oligomeric structures in water, their subunits are effectively cross-linked by TAT.
Activated ethylenes of the general formula CH2d CH-X (X ) CN, COOR, CONH2, COR, NO2, SO2R, etc.) can be used in the chemical modification of protein nucleophilic functional groups. Acrylonitrile (CH2dCHCN) is considered the principal representative of the group (1, 2). Systematic investigation of interactions between acrylonitrile and amino acids and peptides or model compounds have previously been reported by Friedman and his collaborators (2-7), who demonstrated that of the nucleophilic groups of proteins thiol groups are modified most readily. Amino groups are less reactive by 2-3 orders of magnitude, and the imidazole groups of histidine are still less reactive (8). Other functional groups of proteins are not modified. In addition, thiol groups (7, 9, 10) and amino groups (11) of proteins have been demonstrated to be successfully modified by acrylamide (CH2dCH-CONH2), though the reaction is slower (approximately by a factor of 10) than that with acrylonitrile. As a result, β-carbamidoethyl groups (CH2CH2CONH2) are introduced into the protein molecule. These groups closely resemble glutamine residues. Neither protein electric charge nor pI is altered, nor is solubility decreased, a common result after modification by acrylonitrile or alkylating agents (11). Acrylamide therefore is seen as an agent for the “delicate” modification of proteins. Finally, there are also publications on the modification of proteins by several other activated ethylenes: methylacrylate (2, 7), 4-vinylpyridine (2, 12), and alkylvinylsulphones (2, 13, 14). A large variety of N-substituted acrylamides can be synthesized easily (15). Reactivity of their ethylenic bonds in nucleophile-driven addition reactions varies considerably depending on the nature of N-substituents (16). We therefore investigated several bifunctional and * 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.
trifunctional derivatives of acrylamide as potential crosslinking agents for protein lysine residues. 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT)1 was found to be a very promising agent because of its high activity and stability in water solutions. MATERIALS AND METHODS
Chemicals. Human serum albumin from Calbiochem, bovine serum albumin, myoglobin, acrylamide, N,N′diacryloyl-diaminomethane, N-acetyl-L-cysteine, dithiothreitol, and 2,4,6-trinitrobenzene sulfonic acid from Serva, R1-casein from Sigma, and NR-acetyl-L-histidine from Reachim (Russia) were used. Recombinant human interferon-R2b (IFN-R2b), recombinant human interferon-γ (IFN-γ), recombinant tumor necrosis factor R (TNFR), and recombinant human growth hormone (hGH) were products of Biofa AB (Lithuania). Protein markers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from Sigma, Pharmacia, and Bio-Rad were used. 1H-NMR Analysis. 1H-NMR spectra were recorded on a Hitachi 22 spectrometer, operating at 90 MHz, using tetramethylsilane as the internal standard. Synthesis of Cross-Linking Agents. N,N′-Diacryloyl-diamines (Table 1, nos. 3-5). Acryloylchloride (8 mL, 0.10 mol) was slowly added with stirring to a mixture of diamine (0.05 mol), anhydrous sodium acetate (9 g, 0.11 mol), chloroform (50 mL), and hydroquinone (0.05 g) at 10 °C. The reaction mixture was stirred for 2 h at room temperature and then was filtered. The filtrate was left overnight at 4 °C. A white precipitate was collected and 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 (CD31 Abbreviations: TAT, 1,3,5-triacryloyl-hexahydro-s-triazine; IFN-γ, recombinant human interferon-γ; IFN-R2b, recombinant human interferon-R2b; hGH, recombinant human growth hormone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; TNFR, recombinant tumor necrosis factor R.
10.1021/bc9800318 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/25/1998
Cross-Linking of Protein Subunits
Bioconjugate Chem., Vol. 9, No. 6, 1998 745
Table 1. Rate Constants k (M-1 s-1) for Reactions of Potential Cross-Linking Agents with Model N-Nucleophiles in Water Solution at 25 °C N-nucleophile NH
derivative of acrylamide 1 2 3 4 5
acrylamide N,N-diacryloyl-diaminomethane N,N-diacryloyl-1,2-diaminoethane N,N′-diacryloyl-1,3-diaminopropane N,N′-diacryloyl-piperazine
CH2dCHCONH2 CH2dCHCONHCH2NHCOCHdCH2 CH2dCHCONHCH2CH2NHCOCHdCH2 CH2dCHCONH(CH2)3NHCOCHdCH2 COCH
NH2CH2COOk × 105 a
k × 103 a
structure
CH2
34.00 ( 3.5b 16.66 ( 1.43 5.06 ( 0.50 2.79 ( 0.15 29.07 ( 2.37
38.30 ( 2.2b 32.54 ( 2.59 7.53 ( 0.58 5.52 ( 0.54 63.10 ( 2.75
752.00 ( 46.67
769.00 ( 47.33
N N COCH
6
1,3,5-triacryloyl-hexahydro-s-triazine
CH2
CH2
CHCO N
N COCH
CH2
N COCH
7 a
diacrylimide
CH2
CH2dCHCONHCOCHdCH2
6123 ( 201
14590 ( 1300
b
Rate constants for one electrophilic group (CH2dCHCO-) are given. Standard deviations are given.
Scheme 1
Scheme 2
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 for C9H14N2O2: 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): δ 3.76 (s, 8H, piperazine ring), 5.91 (q), 6.31 (q), 7.04 (q, 6H, CHdCH2). 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT, Table 1, no. 6). TAT was prepared as described by T. L. Gresham and T. R. Steadman (19) and recrystallized from water. TAT polymerized when heated and no definite 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 was prepared as previously described (20). The product was purified by column chromatography on silica gel using ethyl acetate/chloroform (1/1, v/v) as the eluent. Diacrylimide 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 reactions shown in Scheme 1 were followed spectrophotometrically in thermostated cell at 255 nm for unsaturated amides no. 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 255 nm 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 coefficient of piperidine at 255 nm was 1.46 M-1 cm-1. UVabsorption spectra of addition products were shifted toward the short-wave side with respect to the spectra of initial unsaturated amides. From 6 to 10 kinetic runs were carried out for each reaction. Molar concentration of the product at the time t(x) was calculated by the equation
x ) a(Ao - A/Ao - Ainf) where A is the absorbance at the time t, Ao is the initial absorbance and Ainf is the final absorbance, when the reaction is completed, a is the initial concentration of N-substituted acrylamide. The kinetics of the reactions shown in Scheme lB were studied in alkaline solutions at several pH values and rate constants were calculated for anion NH2CH2COOas the nucleophile. The kinetics of alkaline hydrolysis of diacrylimide were also investigated spectrophotometrically 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 the concentration of free amino groups in aliquots of the reaction mixture every 30 min over a period of 3-4 h, using the 2,4,6-trinitrobenzene sulfonic acid method (21) as described by Jonusˇiene˘ et al. (22). Three kinetic runs were carried out for each reaction. Second-order rate constants were calculated from the reaction rate values obtained by the method of cubic smoothing splines (23) at the initial moment and after modification of 50% of the amino groups. The kinetics of the reaction of TAT with N-acetyl-Lcysteine thiol groups were followed spectrophotometri-
746 Bioconjugate Chem., Vol. 9, No. 6, 1998
Dienys et al.
Scheme 3a
a
P represents a protein molecule.
Table 2. Rate Constantsa and Half-Lives of Protein Amino Groups Modification by TAT at pH 9.2 and 30 °C protein
c[TAT]o (mM)
k × 103 b (M-1 s-1)
kl × 103 c (M-1 s-1)
half-lifed (h)
Rl-casein BSA hGH IFN-R2b IFN-γ
2.75 3.24 3.69 8.13 8.13
8.58 ( 0.43e 9.15 ( 0.80 7.36 ( 0.58 9.36 ( 0.88 15.11 ( 3.49
6.66 ( 0.71e 4.19 ( 0.18 6.15 ( 0.58 8.06 ( 0.38 8.13 ( 1.62
3.19 ( 0.27e 3.21 ( 0.22 2.73 ( 0.29 0.88 ( 0.05 0.69 ( 0.15
a Rate constants for one electrophilic group (CH dCHCO-) of 2 TAT are given. b Rate constants calculated at the initial time. c Rate constants calculated after modification of 50% of the amino groups. d Time required for modification of 50% of the amino groups. e Standard deviations are given.
cally in a thermostated cell, at 275 nm. Six kinetic runs were carried out for each experiment. The second-order rate constants for the N-acetyl-L-cysteine dianionic form were calculated from initial rate values at 6.5, 10.5, and 14.5 °C: 2.67 ( 0.08 M-l s-1, 3.26 ( 0.22 M-1 s-1 and 3.69 ( 0.51 M-1 s-1, respectively. The rate constant at 25 °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 followed spectrophotometrically in a thermostated cell at 275 nm, under pseudo-first-order conditions (150-fold excess of NR-acetyl-L-histidine). Three kinetic runs were carried out for each experiment. The second-order rate constants calculated 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 °C was 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 amino groups was between 0.06 and 0.59 mM. TAT was added in 4.5-67-fold molar excess with respect to the concentration of amino groups in the protein solution. The reaction mixture was incubated at 30 °C. Aliquots were withdrawn over the course of the reaction, and the reaction was stopped by the addition of excess cysteine or dithiothreitol. Samples of the reaction mixture were dialyzed and analyzed by SDS-PAGE for identification of cross-linked protein forms. SDS-PAGE. SDS-PAGE was carried out on the Mighty Small electrophoresis unit (Hoefer Scientific Instruments) according to the method of Laemmli (24). The acrylamide concentrations used in the gel were 12.5% (for hGH, TNFR), 15% (for INF-γ, myoglobin), and 7.5% (for human serum albumin). Proteins were stained with Coomassie Brilliant Blue R-250 and the gels were scanned at 633 nm using the Dual-Wavelength TLC scanner (CS 930, Shimadzu). RESULTS AND DISCUSSION
Investigation of Cross-Linking Agents. Six difunctional and trifunctional derivatives of acrylamide were synthesized. Their activity in nucleophilic addition reactions was evaluated by measuring the kinetics of the
reactions with low molecular weight model N-nucleophilesspiperidine and the anion of glycine (Scheme 1). The rate constants measured are given in Table 1. For comparison, acrylamide is included together with its polifunctional derivatives. N,N′-Diacryloyl-diaminoalkanes (Table 1, nos. 2-4) were found to be less reactive than acrylamide. They are of little interest as cross-linking agents, however, because their rate of modification is too low for practical purposes. The half-life of the most reactive protein amino groups at 25 °C is over 6 h if a concentration of acrylamide of 0.1 M is used (11). Diacrylimide was the most active of the tested compounds (Table 1, no. 7). It is not, however, stable in alkaline solutions. It is hydrolyzed to form acrylamide and acrylate (25). Diacrylimide is a weak acid, and in alkaline solution it ionizes partly to form the anion (CH2d CHCO)2N:-; a process which needs to be taken into account when the kinetics of its hydrolysis and nucleophilic addition of amines are studied. We measured the diacrylimide ionization constant (pKa ) 10.9) using the spectrophotometric method (26) and investigated the kinetics of its alkaline hydrolysis. The kinetic data are consistent with the mechanism including, as the slow step, the attack of diacrylimide unionized molecule by an hydroxide anion (Scheme 2). The rate constant of the reaction described under Scheme 2 at 25 °C in water solution was 4.35 M-1 s-1, a value similar to or slightly higher than those for the hydrolysis of saturated imides: 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 diacrylimide is approximatly 3 h, at pH 10, approximately 30 min. Diacrylimide may therefore be of interest, under conditions where temporary cross-linking is required. We found that the best cross-linking agent from the compounds listed in Table 1 was TAT. Its activity was conveniently high and its stability in water was remarkable. No change was identifed after storing TAT in buffer, pH 9.2, over the course of 1 week at room temperature. Its three active groups make it an especially effective cross-linking agent. In order to characterize the reactivity of TAT, the kinetics of its reactions with two other model nucleophiles N-acetyl-L-cysteine and NR-acetyl-L-histidine were investigated. The rate constant of the anionic form of N-acetyl-L-cysteine (a model of thiol groups of proteins) was 5.54 M-1 s-1 at 25 °C in water, a value which exceeded, by nearly three orders of magnitude, the rate constant 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 a nucleophile as the amino group. The rate constant of the reaction between NR-acetyl-L-histidine and TAT at 25 °C and pH 9.0 was 1.19 × 10-5 M-1 s-1. Consequently, it is clear that, if there are no free thiol groups, TAT is quite a specific modifying agent for protein amino groups. Modification and Cross-Linking of Proteins. The first step in the interaction between TAT and a protein molecule is generally the addition of an amino group to one of the acryloyl groups (Scheme 3). If free thiol groups
Cross-Linking of Protein Subunits
Bioconjugate Chem., Vol. 9, No. 6, 1998 747
Figure 2. Kinetics of hGH cross-linking by TAT (0.05 M borate buffer, pH 9.2, 30 °C; initial concentrations: hGH, 0.3 mg/mL, 14 µM; TAT, 1.8 mM). Experiment A (4), Zn2+ (28 µM) was added together with TAT; experiment B (O), Zn2+ (28 µM) was added after 25% of amino groups were modified by TAT.
Figure 1. (Top) SDS-PAGE (15%) of IFN-γ samples. Lane 1, protein markers; lane 2, unmodified IFN-γ; lanes 3-5, samples taken at 15, 30, and 60 min. Concentration of IFN-γ, 0.16 mg/ mL; initial concentration of amino groups, 0.18 mM; concentration of TAT, 1.0 mM. (Bottom) SDS-PAGE (12.5%) of hGH samples modified in the absence of Zn2+. Lane 1, unmodified hGH; lanes 2 and 3, samples taken at 60 and 120 min; lane 4, protein markers. Concentration of hGH, 0.75 mg/mL; initial concentration of amino groups, 0.39 mM; concentration of TAT, 4.5 mM. (Middle) SDS-PAGE (12.5%) of hGH samples modified in the presence of Zn2+. Lane 1, unmodified hGH; lanes 2 and 3, samples taken at 60 and 120 min; lane 4, protein markers. Concentrations of hGH, amino groups and of TAT were the same as those in the bottom panel. Molar ratio of Zn2+ to hGH in the reaction mixture was 2:1.
are present, though, their reaction precedes that of the amino groups. Intra- and intersubunit cross-linking of amino groups then follows. We therefore studied the kinetics of the TAT-induced modification of the amino groups of several proteins (Table 2). It was found that the rate constants decreased during the addition reaction. Solubility of TAT in water is limited (approximately 40 mM at room temperature). Half of the amino groups are modified in less than 15 min in the presence of such a concentration of TAT at 30 °C and pH 9.2. We therefore used mixed solvents watermethanol and water-ethanol to promote the modification rate in some cases (27). Cross-linking investigations were carried out with several proteins, both monomeric and oligomeric forms. The course of cross-linking was followed by SDS-
electrophoresis of samples of reaction mixtures. Most experiments were performed at pH 9.2 and 30 °C using a 4.5-14-fold excess of TAT compared with the concentration of amino groups present. At pH 8.0, though, using the maximum possible concentration of TAT (approx. 40 mM) and leaving the reaction mixture overnight at 30 °C practically complete cross-linking could also be achieved. At the protein concentrations used (0.5-2.0 mg/mL), no cross-linking was registered for proteins which were monomeric in water (see, for example, Figure 1, bottom). But after modification by TAT, electrophoretic bands of proteins were slightly displaced and became broader. The change was greatest for the proteins having the greater number of Lys residues (human serum albumin, myoglobin). Proteins, that are dimeric in water [for example, IFN-γ (28)], were transformed by TAT into covalently bonded dimers, which were not separated during SDSelectrophoresis (Figure 1, top). It is known that Zn2+ ions induce the dimerization of hGH (29), an observation confirmed by our measurements of the action of TAT. In the presence of Zn2+, cross-linked dimers were formed (Figure 1, middle), but no dimers were detected in the absence of Zn2+ (Figure 1, bottom). The kinetics of hGH cross-linking are shown in Figure 2. If Zn2+ ions were added after amino groups had been already partly modified by TAT (Figure 2, experiment B), formation of cross-linked dimers occurred more rapidly, where Zn2+ and TAT were added together (Figure 2, experiment A). Apparently, the cross-linking step 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 also investigated (Figure 3). Polymerization was evident after 30 min, when the dimeric form was clear, its presence was maximal after 90 min, but at longer time intervals the trimeric form developed to become predominant, though two bands of molecular weight higher than the dimeric form were identifiable. Most likely, both repre-
748 Bioconjugate Chem., Vol. 9, No. 6, 1998
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, protein markers. Concentration of TNFR, 0.15 mg/mL; initial concentration of amino groups, 0.06 mM; concentration of TAT, 4 mM.
sent cross-linked trimers (31). A very weak band of a molecular weight corresponding to that of a hexamer appeared also at long time intervals. X-ray studies indicate the assembly of two trimers of TNFR to make a hexameric unit in the crystal form (32). Dimerization of trimers may also occur to some extent in solution. Cross-linking of protein amino groups by TAT is a very simple and rapid way to establish whether protein oligomers are formed in solution. The high stability of TAT in water promotes its utility. It would be very difficult to conduct experiments similar to those shown in 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, INFγ, and TNFR. LITERATURE CITED (1) Means, G. E., and Feeney, R. E. (1971) Acrylonitrile. Chemical Modification of Proteins, pp 114-117, Holden-Day Inc., San Francisco. (2) Friedman, M., and Wall, J. S. (1966) Additive linear freeenergy relationships in reaction kinetics of amino groups with R,β-unsaturated compounds. J. Org. Chem. 31, 2888-2894. (3) Friedman, M., and Wall, J. S. (1964) Application of a Hammet-Taft relation kinetics of alkylation of amino acids and peptide model compounds with acrylonitrile. J. Am. Chem. Soc. 86, 3735-3741. (4) Friedman, M., Wall, J. S., and Cavins, J. F. (1965) Relative nucleophilic reactivities of amino groups and mercaptide ions in addition reactions with R,β-unsaturated compounds. J. Am. Chem. Soc. 87, 3672-3682. (5) Friedman, M. (1967) Solvent effects in reactions of amino groups in aminoacids, peptides and proteins with R,βunsaturated compounds. J. Am. Chem. Soc. 89, 4709-4713. (6) Cavins, J. F., and Friedman, M. (1967) New amino acids derived from reactions of epsilon - amino groups in proteins with alpha, beta-unsaturated compounds. Biochemistry 6, 3766-3770. (7) Cavins, J. F., and Friedman, M. (1968) Specific modification of protein sulfhydryl groups with R,β-unsaturated compounds. J. Biol. Chem. 243, 3357-3360. (8) Bosshard, H. R., Jorgensen, K. U., and Humbel, R. E. (1969) Preparation and properties of cyanoethylated insulin. An insulin derivative with blocked amino- and imidazole-groups. Eur. J. Biochem. 9, 353-362.
Dienys et al. (9) Edwards, P. M. (1975) Neurotoxicity of acrylamide and its analogues and effects of these analogues and other agents on acrylamide neuropathy. Br. J. Ind. Med. 32, 31-38. (10) Brune, D. C. (1992) Alkylation of cysteine with acrylamide for protein sequence analysis. Anal. Biochem. 207, 285-290. (11) Danilevicˇiu j te˘ , M., Adome˘ niene˘ , O., and Dienys, G. (1981) Kinetics of interaction of acrylamide with amino groups of some proteins. Org. React. 18, 217-223. (12) Friedman, M., Krull, L. H., and Cavins, J. F. (1970) The chromatographic determination of cystine and cysteine residues in proteins as 5-β-(4-pyridylethyl)cysteine. J. Biol. Chem. 245, 3868-3871. (13) Masri, M. S., and Friedman, M. (1988) Protein reactions with methyl and ethyl vinyl sulfones. J. Protein Chem. 7, 4954. (14) Morpurgo, M., Veronese, F. M., Kachensky D., and Harris, J. M. (1996) Preparation and characterization of poly(ethylene glycol) vinyl sulfone. Bioconjugate Chem. 7, 363-368. (15) Beckwith, A. L. J. (1970) Synthesis of Amides. The Chemistry ofAmides (J. Zabicky, Ed.) pp 73-186, Interscience Publishers, London. (16) Gave˘ nas, G., Dienys, G., and Adome˘ niene˘ , O. (1984) Kinetics of nucleophilic addition of piperidine and glycine to N-substituted and N,N-disubstituted acrylamides. Org. React. 21, 295-303. (17) Hirose, K., Ihashi, Y., and Yoshizava, M. (1966) Infrared spectra of poly(methylenebisacrylamide). Kogyo Kagaku Zaschi 69, 240-244. (18) Danusso, F. (1967) Sintesi di poli-amidi-amine lineari ad elevato peso moleculare. Nota II. Polimeri da diamine disecondari a bisacrilamidi diverse. Chim. Ind. 49, 453-457. (19) Gresham, T. L., and Steadman, T. R. (1949) Triazines from formaldehyde and nitriles. J. Am. Chem. Soc. 71, 1872-1873. (20) Merger, F. (1968) Reaktionen des harnstoffs mit acrylsaure und methacrylsaurechlorid. Chem. Ber. 101, 2419-2425. (21) Satake, K., Okuyama, T., Ohashi, M., and Shinoda, T. (1960) The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-trinitrobenzene-l-sulfonic acid. J. Biochem. 47, 654-657. (22) Jonusˇiene˘ , Z., Dienys, G., and Steponavicˇius, J. (1988) Determination of amino groups in proteins and peptides with 2,4,6-trinitrobenzene sulfonic acid. Zh. Anal. Khim. 43, 536540. (23) Dagys, R., Pauliukonis, A., Kazlauskas, D., Mankevicˇius, M., and Simutis, R. (1986) Determination of initial velocities of enzymic reactions from progress curves. Biochem. J. 237, 821-825. (24) Laemmli, U. K. (1970) Laemmli cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (25) Edward, J. T., and Terry, K. A. (1957) The hydrolysis of amides and related compounds. Part IV. Diacetylamine and succinimide in aqueous alkali. J. Chem. Soc. 3527-3530. (26) Albert, A., and Serjeant, E. P. (1962) Ionization Constants of Acids and Bases, John Wiley, New York. (27) Dienys, G., Strazdiene˘ , V., and Sereikaite˘ , J. (1996) The effect of solvent on the rate of addition of R1-casein amino groups and model nucleophiles to 1,3,5-triacryloyl-hexahydros-triazine. Org. React. 30, 21-23. (28) Ealick, S. E., Cook, W. J., Vijai-Kumar, S., Carson, M., Nagabhushan, T. L., Trotta, P. P., and Bugg, C. E. (1991) Three-dimentional structure of recombinant human interferon-γ. Science 252, 698-702. (29) Cunningham, B. C., Mulkerrin, M. G., and Wells, J. A. (1991) Dimerization of human growth hormone by zinc. Science 253, 545-548. (30) Van Ostade, X., Tavernier, J., and Fiers, W. (1994) Structure-activity studies of human tumor necrosis factors. Protein Eng. 7, 5-22. (31) Horiguchi, T., Miwa, Y., and Shigesada, K. (1997) The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking. J. Mol. Biol. 269, 514-528. (32) Brookhaven Protein Data Bank filename 2TUN.
BC9800318
