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Bioconjugate Chem. 1990, 1, 114-122
A General Method for Highly Selective Cross-Linking of Unprotected Polypeptides via pH-Controlled Modification of N-Terminal a-Amino Groups Ronald Wetzel,*St Roger Halualani, John T. Stults, and Clifford Quan Departments of Protein Chemistry and Biomolecular Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080. Received November 7 , 1989
A method is described for the highly selective modification of the a-amino groups a t the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride a t pH 6.0 occurs with 90-98% selectivity for the a-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the a-amino moiety-of the reagent or of modified peptide-with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N*-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for a-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way. It should also be possible to confidently cross-link small amounts of proteinaceous biological factors, and thus develop affinity matrixes or make antibodies before the polypeptide of interest has been fully purified or structurally characterized.
Although chemical cross-linking of polypeptides, to another molecule or to a matrix, is an important and oftenused step in the biological and biochemical study of peptides and proteins, there is no good general method available which does not require de novo synthesis of the polypeptide. One commonly used approach, for example, is to incorporate into a nonaqueous synthesis of the polypeptide an N- or C-terminal cysteine residue, which can subsequently be utilized as a reactive handle for crosslinking via a disulfide or thioether linkage (I). Such methods suffer from the disadvantage of requiring time and expertise to do the synthesis and, not incidentally, demand prior knowledge of the structure of the peptide of interest. Such a synthetic approach will sometimes present further problems, due to size or other limitations to chemical synthesis, such as the presence of a disulfide bond in the peptide. Mixtures of peptides, as in a protease digest of a protein, require separate synthesis of each peptide even for uses in which a mixture of cross-linked peptides might be adequate or desirable. The requirement of this conventional technolgoy for de novo synthesis for control of the site of attachment is especially unfortunate in the present age of recombinant DNA based methods for polypeptide synthesis. An alternative approach to de novo synthesis, in print Present address: Macromolecular Sciences Department, Smith Kline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 194060939. 1043-1802/90/2901-0 114$02.50/0
ciple, would be to utilize bifunctional cross-linking reagents to modify unprotected polypeptides in the aqueous phase. Although bifunctional cross-linking has been used with considerable practical success on proteins, the inherent lack of specificity of the available methods limits the control and confidence with which they can be used. In theory, it should be possible to selectively modify the N-terminus of an unprotected peptide in aqueous solution by exploiting the differences between the pK, values of its a-amino group (about 8.0) and the side chain amino groups of Lys (about 10.5) and Arg (about 12) (2, 3 ) . This is particularly attractive since modification of the chain terminus should minimize interference of the cross-link with the amino acid side chains and their content of biological information. Such a modification should fulfill two requirements. First, the ratio of reactivities of the a-amino group to the amino groups of the side chains of Lys and Arg should be as high as possible. Second, the reactivity of the a-amino groups of different peptides should be independent of peptide sequence and composition, so that reaction conditions don’t have to be customized for each peptide. Stark ( 4 ) has shown that the reactivity of cyanate in the neutral pH range with a series of amino acids and short peptides exhibits a very good correlation with the pK, values of their amino groups. This study showed that the relative reactivity between the a-amino group and the t-amino group of AlaLys was based on both their pK, difference and on the relative reaction rates of other model compounds. Stark concluded that a-amino groups
0 1990 American Chemical Society
Selective Cross-Linking of Polypeptides
Table I. SelectivityP*2of 2-IT for a-Amino Groups of N-Terminal Amino Acids
corrected rate, selectivity peptide Adlplmin ratio” AlaGly 0.027 4.5 0.036 6 AspGly 0.066 11 GluAla 0.174 29 GlyGly HisGly 0.159 27 IleAsn 0.036 6 0.024 4 LeuGly MetGly 0.063 11 PheGly 0.038 6 0.029 5 ProGly 0.085 14 SerGly TrpGly 0.081 14 TyrGly 0.061 10 ValGly 0.013 2 AcGlyLysOCH, 0.006 (1) Selectivity ratio is the rate of reaction of the cu-amino group divided by the rate of reaction of the c-amino group of lysine, as measured using the peptide AcGlyLysOCH,.
of peptides should react about 100 times faster than the c-amino group of Lys side chains when reacted a t a pH a t least 1 unit below the lower pK,. This study was restricted to only three N-terminal amino acids (Thr, Ala, and Gly), all of which have relatively small side chains. In contrast to this suggestion of uniform reactivity of peptide a-amino groups, Hunter and Ludwig (5) showed that, in reaction with methyl benzimidate, Gly reacts about 7 times faster than Phe at pH 9.5; taking into account their slightly different pK, values, Gly was calculated to react about 12 times faster than Phe. The authors suggested that the different reactivities of the unprotonated forms of these amino acids derived from differences in steric access of reagent and/or in nucleophilicity. The authors also suggested that it was this inherent reactivity difference which accounts for observations (57) that reaction of insulin with amine-directed reagents generally leads to modification of the A chain terminus (Gly) but not the B chain terminus (Phe). These differential reactivities, not noted when insulin is reacted with cyanate (8), had previously been ascribed to inaccessibility of the B chain N-terminal Phe in the insulin tertiary structure. Thus it appears that selectivity for the cyamino group in the presence of other amino groups can vary both with the reagent and with the nature of the side chain on the N-terminal residue. Selective modification of a-amino groups by pH control has also been reported for other acylating agents, such as aryl isothiocyanates (9). Most attempts to selectively react the N-termini of polypeptides, however, have been with acetic anhydride. For example, reaction of the peptide hormone glucagon with acetic anhydride a t pH 5.5 generates two products, 70% acetylated only on the a-amino group of the N-terminal His residue and 30% diacetylated a t the N-terminus plus the €-amino group of Lysl2 (10). Reaction of the 28 amino acid peptide desacetylthymosin cy1 with acetic anyhydride gives the N-terminally acetylated peptide as about 90% of the product, even though the peptide contains four Lys residues (11). Larger polypeptides may be more difficult to selectively modify. Magee et al. (12) found that reaction with acetic anhydride exhibited little if any selectivity for the N-terminus of trypsinogen; a strategy of reversible protection of Lys residues was required to selectively modify the N-terminus. In this paper we show that for two chemical groups widely used in the cross-linking of peptides, N-hy-
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droxysuccinimide (NHS)l activated carboxylic acids and 2-iminothiolane (2-IT),pH-controlled selectivity’ for a peptide’s N-terminus is relatively poor. Furthermore, at least for 2-IT reactions, we show that peptide reactivities vary considerably with the nature of the N-terminal amino acid. We show that much better results can be obtained using a two-step procedure exploiting the high selectivity for cy-amino groups of acid anhydrides. This simple method can be used to prepare peptides for crosslinking, in very good yields, from already existing peptides, and thus should be a useful general alternative to de novo peptide synthesis. In addition, the method may have some unique applications in modifying mixtures of peptides. EXPERIMENTAL PROCEDURES
Materials. A series of 20 test hexapeptides were synthesized by building the pentapeptide sequence GAKQA on a solid phase, dividing the resin into 20 portions, and completing the synthesis with different N-terminal amino acids. After HF cleavage and deprotection, the peptides were purified by high-performance liquid chromatography (HPLC), lyophilized, and resuspended in water. Purity was assessed by analytical HPLC in two chromatographic systems. Identity was confirmed by mass spectrometry. Other peptides were also chemically synthesized, with the exception of Na-desacetylthymosin a1 [which was prepared by recombinant DNA methods (11)] and its trypsin fragments. Murine interferongamma (13) was provided by L. E. Burton (Genentech). Dipeptides used in this study described in Table I were purchased from Sigma, as was 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB). NHS, sulfosuccinimidyl acetate, 2IT, and dicyclohexylcarbodiimide (DCC) were purchased from Pierce. Iodoacetic anhydride was purchased from Aldrich. Methods. HPLC was performed on a Waters gradient system composed of Model 510 pumps and an automated gradient controller. Separations were on Vydak 5-pm (3-18 reverse-phase columns. Two buffer systems were used, both employing Milli-Q-purified water as solvent A and acetonitrile as solvent B. In system I, 0.1% trifluoroacetic acid (Pierce) was included in both A and Abbreviations: DCC, dicyclohexylcarbodiimide; DTNB, 5,5’dithiobis(2-nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; FAB, fast atom bombardment; HPLC, high-performance liquid chromatography; 2-IT, 2-iminothiolane; MES, 2(N-morpho1ino)ethanesulfonicacid; MS, mass spectrometer; NaOAc, sodium acetate; NHS, N-hydroxysuccinimide. For comparison of reagents we devised a “selectivity” value which would allow us to compare the qualities of reagents such as 2-IT, for which kinetic measurements can be made, with reagents such as acid anhydrides, where multiple additions of reagents are required, reactions are fast, and peptide and solvent compete for reagent. For the first class, selectivity is the ratio of the rate of reaction of the a-amino group to the rate for the c-amino group. For the second class, selectivity is the ratio of all product molecules containing modified a-amino groups to those containing c-amino groups (in each case regardless of whether or not they are also modified elsewhere); where uncharacterized side products exist, they are included in the denominator. In this latter method the selectivity clearly depends on how far the reaction is pushed; values will more closely reflect relative reaction rates if they are assessed on reaction mixtures containing substantial amounts of unreacted starting material. On the other hand, the object of this study is to devise an efficient synthetic strategy, so selectivity under practical conditions is also important. This is why there are two selectivities listed in Table 11.
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B. In system 11, 5 mM heptafluorobutyric acid (Pierce) was included in both. System I was useful for initially identifying products, but for some peptides it could not be used to follow the progress of the reaction because iodoacetate coelutes with the peptide starting material. System I1 allowed us to follow the disappearance of starting material, except that with some peptides the disubstituted product co-elutes with starting material and thus cannot be quantified. Prep collection of modified peptides was performed with the TFA system, followed by lyophilization and resuspension in water. Iodoacetyl peptides were stable for at least 1 year stored at -20 "C. Mass spectra were obtained with a JEOL HXllOHF/ HXllOHF tandem double-focusing mass spectrometer (MS), operating with a JEOL DA-5000 data system. Ionization by fast atom bombardment (FAB) with a 6 keV xenon beam produced positive ions that were analyzed at 10 keV accelerating voltage, 3000 resolution. Peptide products, purified by HPLC, were dried in vacuo and redissolved in water to a concentration of about 1 nmol/ yL. One microliter of this solution was added to 0.5 yL of nitrobenzyl alcohol on the probe tip and loaded into the MS. Nitrobenzyl alcohol was preferred as the matrix because the haloacetyl peptides did not produce intact MH+ ions in glycerol, thioglycerol, or a 5:l ratio of dithiothreitol/dithioerythritol.Conventional FAB spectra were produced by scanning MS-1 and acquiring data at detector 1. Daughter spectra were produced by selecting the 12C isotope peak of the MH+ ions with MS-1, passing it into a collision cell containing a pressure of helium sufficient to attenuate the parent ion beam to 25% of its original intensity, and measuring the MS-2 the fragmentation resulting from collision-induced dissociation. Relative reaction rates of peptides with 2-IT were determined colorimetrically. For the dipeptide study summarized in Table I, peptides (3 mM) were mixed with 10 mM DTNB in 0.4 M sodium acetate (NaOAc), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 5.4. A fresh stock solution of 100 mM 2-IT was prepared in the same buffer and held at 0 "C. This stock solution was diluted 10-fold into the 0 "C reaction mixture to give 10 mM 2IT; the reaction was followed by generation of absorbance at 412 nm. A stock solution of iodoacetic acid anhydride was prepared as follows. A fresh bottle of iodoacetic anhydride (Aldrich) was dissolved in its entirety in dry tetrahydrofuran (THF) to a concentration of 300-400 mM and stored at -20 "C as aliquots in 1.5-mL Eppendorf tubes, which in turn were stored in 50-mL plastic, screw-top, conical tubes containing Drierite and sealed with Parafilm. Such tubes were not opened until equilibrated to room temperature. Stored in this way, reagents were stable for at least 9 months. This procedure was deemed necessary since in our hands iodoacetic anhydride proved unstable in its solid form under conditions in which portions of the stored solid were periodically removed. Reaction of peptides with acid anhydrides was performed as follows. To a solution of the peptide (0.25-1 mM) in aqueous buffer a t 0 "C in a 1.5-mL polypropylene Eppendorf tube was added 1/100 volume of an approximately 300 mM solution of acid anhydride in dry THF. The sample was immediately vortexed. After 3 min, in which time reagent was depleted by a combination of reaction with peptide and hydrolysis, a fresh aliquot of anhydride in T H F was added and the mixture was vortexed. This procedure was repeated until the reaction reached the desired level of completion, usually two
Wetzel et al.
or three additions. Except where indicated in the text, reactions were done a t pH 6.0 in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES). Reactions were analyzed by adding a 10-20-pL aliquot of reaction mixture to 180-190 pL of HPLC buffer A. After vortexing, the solution was centrifuged (Eppendorf) and the supernatant was injected or transferred to vials for HPLC analysis. The NHS ester of iodoacetic acid was prepared as follows. To a mixture of 6.4 g (34.4 mmol) of the free acid of iodoacetic acid and 3.9 g (33.9 mmol) of NHS in 120 mL of dried (3A molecular sieves) T H F at room temperature was added, with stirring, a solution of 7.1 g (34.5 mmol) of DCC in 20 mL of dry T H F over a 3-5 min period. The reaction mixture was stirred overnight with exclusion of light and then filtered and the precipitated dicyclohexylurea was washed with 20 mL of dry THF, which was added to the filtrate. The filtrate was reduced in volume on a rotary evaporator at about 40 "C to about 40 mL, at which point a rapid crystallization occurred. After a few hours at 4 "C the crystals were collected and recrystallized from about 70 mL of ethyl acetate/ acetone by dissolving a t 50 "C and allowing to cool and stand at room temperature overnight. About 940 mg of material was obtained from the crystals obtained and another 1g was obtained from crystallization of the dried filtrate of the first crystallization, for a total yield of 20%. The twice-crystallized material was characterized by MS as the NHS ester of iodoacetic acid. This was stored at 4 "C over CaC1,. Reaction with murine interferon-gamma was accomplished as follows. Five milligrams of the protein in 7 mL of pH 7.5 buffer was brought to 1 mM EDTA and 1 mM DTT and incubated for 1 h at room temperature. The resulting solution was dialyzed with two buffer changes over a period of 24 h against 100 mL of 10 mM NaOAc, 0.25 mM EDTA, pH 5.4. Portions (700 pL) of this solution were mixed with 80 pL of 1M tris-HC1,lO mM EDTA, pH 8 and with 70 pL of an aqueous stock solution (1-5 mM) of iodoacetyl peptide and the reaction mixture was incubated for 20 h at 37 "C. RESULTS
Table I lists selectivity ratios, indicating the preference for reaction at the N-terminal a-amino group to reaction at a Lys €-amino group, for the reaction of 2-IT with a series of dipeptides with varying N-terminal residues. These ratios were calculated by dividing the rate of reaction of the dipeptide, determined colorimetrically as described in the methods, by the similarly determined rate for the dipeptide AcGlyLysOCH,. When 2-IT reacts with an amino group, a sulfhydryl group is generated. Absorbance at 412 nm is developed in the assay by reaction of these released sulfhydryl groups with DTNB. Although the reaction of thiols with DTNB, just as the reaction of 2-IT with amino groups, is relatively slow at pH 5.7, we included a large concentration of DTNB to insure that this reporting reaction was fast compared to the reaction of amino groups with 2-IT. The table shows that in each case reaction at the a-amino group is favored over that of the €-amino group, but that selectivity varies from a high of 29 for GlyGly to a low of 2 for ValGly, with most values falling in the range 4-14. Figure 1 shows the result of reaction of the peptide FGAKQA with 2-IT at pH 5.8. Panel A shows the unreacted peptide. Panel B shows that, with approximately 26% of the peptide as yet unreacted, the reaction product is a mixture containing one major component and several minor ones. The main product, eluting at about
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Selective Cross-Linking of Polypeptides
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Figure 1. Reaction of FGAKQA with 2-IT. Panel A shows unreacted starting peptide (a). Panel B shows the mixture obtained after reaction of 0.4 mg/mL (0.62 mM) peptide with 10 mM 2-IT in 1 M NaOAc, 5 mM EDTA, pH 5.8, for 10 h at room temperature. The peak at 18 min is starting peptide (a). In the reaction mixture, none of the peaks eluting after 20 min are found in a mock reaction without peptide, and thus are assumed to be modified peptide. Products were not characterized. It can be seen that the main peptide product (b) has significant absorbance at 280 nm, the trace for which is offset about 3 min after the 214-nm trace. The chromatographic system is the same as that described in the legend to Figure 2. 22 min, represents 80% of the total reacted peptide (the peak a t 14 min is associated with the reagent). This gives a selectivity ratio' [(monosubstituted + disubstituted/ disubstituted) = (total reacted a t a-amino/total reacted a t t-amino)] of 100/20 = 5. This ratio is in good agreement with the value of 6 determined from the reactions of the dipeptides PheGly and AcGlyLysOCH, with 2-IT (Table I), suggesting that this colorimetric reaction with dipeptides can be considered diagnostic of the general reactivity of peptides with 2-IT. Figure 2 shows the reaction of FGAKQA with two reagents which transfer an acetyl moiety. Panel A shows the reaction mixture with acetic anhydride (two additions of reagent; see the methods) a t p H 6.0; with only 13% of the peptide left unreacted, 98% of what has reacted is acetylated on the a-amino group (peak b) and 2% is acetylated on both the a-amino group and on the tamino group of lysine (peak c; structure confirmed by MS analysis). This gives a selectivity ratio of 100/2 = 50. Panel B shows the reaction with 4 mM sulfosuccinimidyl acetate in 0.1 M MES a t room temperature for 1 h; with 15% of the peptide unreacted, 86% of the product mixture is only a-acetylated, while 14% is also reacted on the lysine side chain. This gives a selectivity ratio of 100/14 = 7, which is comparable to the selectivity of 56 (see above) observed for reactions of N-terminal Phe peptides with 2-IT and is a factor of 7 less specific than the reaction of FGAKQA with acetic anhydride. Panel C shows the result of reacting iodoacetic anhydride with FGAKQA in 1.6 M NaOAc, p H 5.8; 43% of the product is N"-acetyl-FGAKQA, presumably derived from reaction with a mixed anhydride generated by rapid interchange of iodoacetic anhydride with the acetate buffer. Figure 3 shows selected time points from a typical reac-
0
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Figure 2. Formation of Ne-acetyl-FGAKQA by acid anhydride reactions. FGAKQA (500 pL, 0.4 mg/mL, 0.64 mM) was reacted under the following conditions. Panel A: 0.1 M MES buffer, pH 6.0, at 0 "C, three additions of 5 p L each of -350 mM acetic anhydride in THF. The small peak (a) at about 18 min is unreacted starting material, and the large peak (b) at about 22 min is the a-acetylated product. Panel B: 0.1 M MES buffer, pH 6.0, at room temperature, addition of sulfosuccinimidyl acetate to 4 mM and reaction for 1h. The new peak (c) at about 25 min is the a,ediacetylated peptide. Panel C: 1 M NaOAc, pH 5.8, at 0 "C, three additions of 5 p L each of -350 mM iodoacetic anhydride in THF. Coinjection with the reaction mixture from panel A confirms the identity of the 18- and 22-min peaks as unreacted (a) and a-acetylated (b) peptides. The peak (d) at 27 min is the a-iodoacetylatedpeptide and the peak (e) at 34 min is derived from the anhydride reagent. Samples were analyzed as described in the Experimental Procedures with a l%/min gradient of acetonitrile in 0.1% trifluoroacetic acid with detection at 214 nm, full scale = 0.2 AU. tion of iodoacetic anhydride with the hexapeptide SGAKQA in MES buffer. The figure shows the gradual conversion of the peak associated with starting peptide (peak b) to a main later-eluting peak (peak c), which was determined by MS analysis to be iodoacetylated on the N-terminus. After several reagent additions, a second product (peak a) appears; this was shown to be the diiodoacetylated product from reaction of the main product a t the Lys €-aminogroup. Both peaks exhibit absorbance a t 280 nm, in contrast to the starting peptide, consistent with the addition of the iodoacetyl moiety (Ama for iodoacetic acid is 273 nm with log t = 2.66). The peaks eluting a t 10 and 43 min were shown to be associated with the reagent. A series of 20 peptides of sequence X-GAKQA was synthesized in which X = each of the 20 standard biosynthetic amino acids, purified by HPLC and confirmed by MS analysis. Reaction of each peptide with iodoacetic anhydride was monitored by HPLC. Reactions were followed by both gradient systems described in the methods. For some peptides, reactions were quantified after two reagent additions in order to determine the selectivity ratio under favorable circumstances, i.e., with significant amounts (about 20%) of unreacted peptide present. In addition, all peptides were reacted with three reagent additions, conditions more useful synthetically, in which >95% of starting material is converted to products. The
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Table 11. Selectivitya,’ of Iodoacetic Anhydride for a-Amino Groups of the Peptide X-GAKQA three reagent additions
1 Additions *2SO 0
214
X
Ala
214
1
214
2
Arg
1
k3
Asn ASP CysSSCH,CH,OH Glu Gln GlY
4
11
I!
0
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20 30 Fraction Number
40
Figure 3. Progress of the reaction of iodoacetic anhydride with the peptide SGAKQA. Conditions are those described in the legend to Figure 2A. Before addition of reagent only one peak (b), for the starting peptide, is apparent in the A,,, trace of HPLC fractionation of the reaction mixture. After one addition of reagent this peak is diminished to about 30% of its original height, and a new, earlier-eluting peak (c), corresponding to N-a-iodoacetylated peptide, is observed. After three additions only a small amount of starting material is left and the bulk of the product is monoacylated, but there is a third peak (a) eluting after starting material which is the a,€-diacylated peptide. After four additions there is no starting peptide remaining. The Azso trace of the reaction mix after four additions shows that both product peaks absorb at 280 nm as expected (in this chromatogram, the A , trace leads the AZl4 trace by about 3 min). The peaks a t a t o u t 10 min and about 43 min are related to the iodoacetic anhydride reagent.
results of these measurements are listed in Table 11. Percent yields of mono- and diacylated products were calculated as percentages of total product peptide absorbance; where percentages do not sum to 100, it indicates the corresponding amount of uncharacterized other peaks in the chromatogram. The table shows that, when reactions are not driven to complete loss of starting material, one obtains a range of selectivity ratios from 9 to 5 5 , with most values at 18 or above. For each of the 20 peptides, a reaction with iodoacetic anhydride was also conducted at pH 9 in sodium borate buffer. As expected, this gave a much greater amount of peptide product acylated on both the a- and the 6 amino groups and was useful in verifying the elution positions of the diacylated product in the pH 6.0 reaction mixtures. In some cases (indicated in Table 11) reaction products were confirmed by tandem mass spectrometry (MS-MS) analysis. In this way it was possible to not only verify the molecular weights of the mono- and diiodoacetylated products of the Ala peptide but also to assign the acylation positions within the peptide product. Using MS-MS, the reaction products for the His and Met peptides, which theoretically could be alkylated on their side chains by iodoacetate, were confirmed to be unreacted on the side chains. In addition, the acylation position of the Pro peptide, whose aamino group is a secondary amine and therefore of higher pK,, was also confirmed by MS analysis to be the N-terminal a-amino group.
His Ile Leu LYS Met Phe Pro Ser Thr TrP TYr Val
% 70 two a-iodo- diiodoadditions: acetyl acetyl selectivity” selectivity” 87‘ lob 10 18 86 5 15 82 6 81 3 28 30 87 3 79 7 12 17 18 81 5 18 83 5 4 16 70b 23b 76 10 9 15 81 6 8 886 126 33 40 97b 3b 77 3 27 33 26 37 74’ 36 77 13 7 55 86 6 15 50 11 23 80 8 4 9 66 21 8 18 78 11
a Selectivity ratio is the sum of the mono- and diiodoacetylated peptide divided by the diiodoacetylated, which corresponds to the total a-iodoacetyl groups divided by the eiodoacetyl groups. Structure verified by mass spectrometry.
Figure 4 shows the reactions of the peptide FGAKQA with reagents which transfer the iodoacetyl moiety, and thus is analogous to Figure 2C. Panel A shows the product (peak b), after two reagent additions, with iodoacetic anhydride. In this HPLC system the disubstituted product appears as a trailing shoulder on a peak (peak c) late in the chromatogram associated with reagent (as confirmed by panel B). The relative amounts of monoand disubstituted products can be seen in the A,,, trace. Adjusting the peak height of the disubstituted product to account for its content of two chromophores (using the factor 0.65 derived from analysis of the chromatogram in panel C), the product distribution when 11%of the starting material remains unreacted is 97% monosubstituted and 3% disubstituted, for a selectivity ratio of 33 (this number was confirmed by a similar analysis of another reaction mixture analyzed in HPLC system 11, in which both reaction products can be quantified in the A,,, trace). Panel C shows the reaction mixture with the NHS ester of iodoacetic acid, and the coinjection experiment shown in panel D confirms that the main products of this reaction are identical with the mono- (26 min) and disubstituted (33 min; see A,,, trace) products with iodoacetic anhydride. The reaction mixture is qualitatively different, however, in the appearance of a new product (not characterized but not associated with reagent) a t 23 min, as well as a much greater amount of disubstituted product (33 min). Even with the exclusion of the unidentified product, the selectivity ratio favoring asubstitution in this reaction is only 2, under conditions in which the extent of reaction, as judged by the 11% remaining unreacted peptide, is identical with the reaction in panel A. Thus for the iodoacetyl group, one sees an improvement in selectivity of a factor of 33/2 = 16.5 comparing the acid anhydride to the NHS ester, an enhancement even better than that seen in the acetyl series (Figure 2). Figure 5 shows the product of the reaction of a series of N“-iodoacetyl-peptides with murine interferongamma, which contains a Cys residue at its C-terminus. The reaction is incomplete, even after a second cycle of
Bioconjugate Chem., Vol. 1, No. 2, 1990
Selective Cross-Linking of Polypeptides
I I c.
I A.
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5
6
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Figure 5. 12.5% SDS polyacrylamide gel on mixtures of reac-
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Figure 4. Iodoacetylation of FGAKQA. Panel A shows the
reaction mixture of the peptide with iodoacetic anhydride under the reaction and chromatographic conditions described in the legend to Figure 2A, except that reaction was terminated after two reagent additions. Peak a is unreacted starting material and peak b is the N-terminally acylated product. The A,,, peak at about 34 min in panel A is predominantlyderived from iodoacetic anhydride, as seen in panel B, a mock reaction mixture without peptide. However this peak contains a small trailing shoulder (c) which is the a,c-diiodoacetyl-FGAKQA. Quantitation of this species is possible since it, but not the iodoacetic acid related material, also absorbs at 280 nm. Panel C shows the reaction mixture of 4 mM of the NHS ester of iodoacetic acid with FGAKQA at 25 "C after 1 h. The peak (a) at about 17 min is unreacted peptide, the peaks at 26 and 33 min are the cy-mono- (b) and cy ,e-diodoacetylated (c) peptides, respectively, and the peak (d) at 23 min is unidentified but not seen in a mock reaction without peptide and so is presumed to be another peptide side product. As compared to iodoacetic anhydride, this reagent does not produce the decompositionproduct migrating at around 34 min. Panel D shows a coinjection of the reaction mixtures used to generate panels A and C. Note that all three products exhibit A280. The A214trace leads the A,, trace by 2-3 min. reaction with fresh peptide; since precautions were taken to assure that the interferon Cys was fully reduced and available for reaction (see the Methods) incomplete reaction suggests that some of the molecules do not contain a C-terminal Cys. In fact, some preparations of this protein lack C-terminal residues due to a difficult to control limited proteolysis. The gel shows that the only product of each reaction is a discrete higher MW protein, whose migration retardation correlates with the MW of the reacting peptide. In a control experiment, human interferongamma, which contains no Cys, was shown to not react with N"-iodoacetyl-peptide (data not shown). DISCUSSION In attempts to devise a general, highly selective crosslinking scheme for peptides, we initially investigated the use of 2-IT at a pH of 6 or below. However, as Table I and Figure 1 show, the selectivity of this reagent varies significantly depending on the N-terminal amino acid and for most amino acids is substantially lower than the factor of 100 found by Stark for the cyanate reaction ( 4 ) . Since we had previously observed (11) a high selectivity
tion of a series of Nu-iodoacetyl-peptideswith murine interferon-gamma (see the Methods). Aliquots of reaction mixtures were mixed with SDS-gelloading buffer containing 10 mM iodoacetamide, heat-denatured, and loaded onto the gel. The finished gel was stained with Coomassie Brilliant Blue. Lane 1is unreacted interferon, and lane 2 shows the reaction product with iodoacetic acid. The other lanes show reaction with these iodoacetyl-peptides, all prep collected from HPLC before reaction: GRTQRLQTLTNLF (lane 3), SRVSQRGRLTLNLESGRWR (lane 4), norleucine-FPTIPLSR (lane 5), EVVEEAEN (lane 6), SDAAVDTSSEITTK (lane 7), SDAAVDTSSEITTKDLKEKKEVVEEAEN (lane 8). The last peptide is N"desacetylthymosina,,and the previous two are its main trypsin digestion products. in the acetylation of the a-amino group of desacetylthymosin al, a 28 amino acid peptide containing four Lys residues, it seemed that acid anhydrides might in general achieve selectivities more like that of cyanate. If true, reaction of a peptide with the anhydride of an ahaloacetic acid would generate an N-terminally derivatized intermediate which could subsequently be reacted with sulfhydryl groups of other molecules or matrices. To demonstrate that this strategy would be useful, we set about to address the following questions: (a) What are the selectivities for the reaction of iodoacetic anhydride for peptide a-amino groups and how do they compare to values for other bifunctional reagents, including other means of introducing the a-iodoacetyl moiety? (b) Is protection/ deprotection of Cys residues feasible and are there any other potential side reactions of the a-iodoacetyl moiety with amino acid side chains? (c) Is the a-iodoacetamido moiety on a peptide sufficiently reactive to modify a sulfhydryl group on a macromolecule or solid support? Selectivities? Table I1 shows that selectivitiesfor reaction of iodoacetic anhydride with a series of hexapeptides containing a competing Lys range from 9 to 55, substantially better than the 2-IT reaction (Table I and Figure 1)in degree of preference for the a-amino group and in uniformity with respect to N-terminal amino acids. Analysis of the data in Figures 2 and 4 (see the Results) suggests that a-amino group selectivities of acid anhydrides can be 10-20-fold higher than those of NHS-activated esters of the same acyl groups. Thus, 2-IT and NHS esters seem to behave relatively unselectively, while acid anhydrides are more highly selective for a-amino groups and discriminate less on the basis of N-terminal amino acid identity, qualities similar to those of cyanate (4). For the acid anhydride reaction, the bulkiness of the
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side chain of the N-terminal amino acid does not seem to greatly influence selectivity. Although the lowest observed selectivity of iodoacetic anhydride is for N-terminal Tyr, the values for the equally bulky Phe and Trp are much better. In fact, the low selectivity of Tyr probably derives not from its bulk but from its chemical nature. Under conditions designed to convert essentially all of the starting material (three reagent additions at pH 61, the Tyr and His peptides yield the lowest selectivity for the a-amino group (Table 11). In contrast, His in a dipeptide exhibits high selectivity for the a-amino group when reacted with 2-IT (Table I). Examination of the course of the reactions of iodoacetic anhydride with HGAKQA and YGAKQA reveals that the reduced ratio of a-amino reactivity to t-amino reactivity is not due to a decreased rate of reaction of the a-amino group but rather to a n increased rate of reaction of t h e t-amino group of Lys. This suggests that the His and Tyr residues of these peptides can catalyze the iodoacetylation of the Lys residues. This makes sense chemically, since both residues would be expected to undergo acylation on their side chains to yield unstable intermediate capable of transferring the acyl moiety to another group. In fact, a similar intramolecular interaction between the Tyr and His residues of angiotensin I1 has been postulated on the basis of the acetylation and deacetylation kinetics of these groups (14). This interpretation suggests that Tyr and His residues at other positions within a peptide may have similar effects. These catalytic transfers must be occurring intramolecularly, since the presence of HGAKQA in a mixed peptide reaction mixture does not alter the ale product ratio of FGAKQA with iodoacetic anhydride (data not shown). The relatively poor selectivity found by Desbuguois (10) for the acetylation of glucagon (see above) may thus in part be due to the presence of the His residue at the N-terminus of this peptide. The above discussion suggests that selectivities may vary with amino acid composition of peptides. However, our studies suggest that with any particular peptide results will be better with acid anhydrides than with many other reagents. Further, reactions with a series of peptides containing up to four Lys or five Arg, for alkylation of murine interferon-gamma (Figure 5), gave the high selectivities expected from the number and pK, values of their basic groups (data not shown), suggesting that one can reasonably expect most peptides to behave qualitatively as those described here. Reactions of Amino Acid Side Chains. Three of the 20 amino acids normally used in ribosomal peptide synthesis, Cys, Met, and His, might be readily alkylated by the a-iodocarboxyl group, while the amino groups of Lys and the N-terminus are also reactive but require more stringent conditions ( 3 ) . By incubation of our test peptides XGAKQA containing N-terminal His, Met, and Gly with iodoacetate under planned reaction conditions, we observed no detectable alkylation (data not shown). Furthermore, the reaction products of these peptides with iodoacetic anhydride were characterized by MS analysis and found to be iodoacetylated on the a-amino groups but not alkylated by a N*-iodoacetamido moiety or by iodoacetate (Table 11). Since Cys residues of the peptide would almost certainly be alkylated under our reaction conditions, one must incorporate a Cys protection step into the procedure for peptides containing free Cys residues. Prior reaction of a Cys-containing peptide with a disulfide such as oxidized 2-mercaptoethanol or DTNB converts the Cys into a mixed disulfide incapable of being alkylated by
Wetzel et al.
iodoacetate under reaction conditions (Table II), and the free sulfhydryl of the Cys can be regenerated by mild reduction after the iodoacetylation and subsequent crosslinking are completed (R.H. and R.W., unpublished). Reaction with Sulfhydryl Groups. Although we have not characterized the reaction of Na-iodoacetyl-peptides with thiol compounds in detail, we have conducted reactions with a number of different mercaptans. For example, reaction of 0.16 mM Ne-iodoacetyl-FGAKQA with 0.8 mM thiocholine in 0.1 M Tris HC1, 2 mM EDTA, pH 8.5 at 37 "C, goes to completion within 1 h. Complete reactions have also been achieved with cysteine, glutathione, and 0-mercaptoethanol (data not shown). Finally, Figure 5 shows that a series of iodoacetyl-peptides varying considerably in size and amino acid composition can be efficiently attached to a sulfhydryl group of a macromolecule, in this case the Cys of murine interferongamma (13). Practical Considerations. Acetylation of proteins has traditionally been done in NaOAc buffer at high concentration, because this buffer catalyzes the hydrolysis of the 0-acetyltyrosine also formed from reaction with acetic anhydride (15). However, we found that these conditions gave poor results, for two reasons. First, use of 1 M NaOAc led to a mixture of iodoacetylated and acetylated peptides, presumably due to rapid generation of mixed acid anhydrides by reaction of acetate buffer with iodoacetic anhydride. Thus, when we reacted peptides with iodoacetic anhydride in 1.0 M. NaOAc, pH 6.0, about half of the product formed was the peptide N-terminally modified with the acetyl, rather than a-iodoacetyl, group (Figure 1). Second, we found that high concentrations of acetate at pH 6 are so weakly buffering that the acid generated from hydrolysis of the anhydride reagent quickly lowers the pH to a point where no further reaction of amino groups with acid anhydride takes place; thus the standard acetate buffer is a poor choice even for reactions with acetic anhydride, since much more anhydride must be added to get a reasonable extent of reaction. For these reasons we chose as a buffer MES, a sulfonic acid derivative which buffers well a t pH 6.0 but is presumably too weakly nucleophilic to form significant amounts of mixed anhydride. Comparison of Figures 2C and 4A shows that MES buffer at pH 6 not only avoids the formation of acetylated side products in reactions with iodoacetic anhydride, but it also promotes the conversion of starting material much better than NaOAc a t pH 6. Although we have not observed 0-acetylation to be a problem in MES buffer, it would be simple to reverse this side reaction, were it to occur, by a subsequent incubation in concentrated acetate or another reagent (15). In some cases it may be advantageous to carry out the iodoacetylation reaction in the presence of a protein denaturant. Reaction of FGAKQA with iodoacetic anhydride in 0.1 M MES, pH 6, in the presence of 5 M guanidine hydrochloride or 7 M urea, under conditions in which about 10% of the starting peptide is unreacted, gives a selectivity ratio of about 20 (data not shown). For the urea reaction, this extent of reaction was achieved with two reagent additions, each 1%of the reaction volume of -350 mM anhydride (Le., the usual conditions). For the guanidine hydrochloride reaction, two additions of 0.5% were required; two additions of 1% reagent gave a reaction mixture entirely depleted in starting material and enhanced in diacylated peptide (thus giving lower selectivity). Presumably guanidine hydrochloride either slightly enhances the acetylation kinetics or inhibits the kinetics of hydrolytic breakdown of the reagent.
Selective Cross-Linking of Polypeptides
In general, reaction conditions, once optimized, can be used with different polypeptides and with some variation in other conditions. For best results, however, it is suggested that reagent excess, etc., be optimized on a test peptide for a given set of conditions. Use of anhydride from different sources or use of different buffers, pH conditions, or other solvent additives (like guanidine hydrochloride) may effect the extent to which a given amount of reagent acylates the polypeptide. Although the reaction conditions are surprisingly tolerant of changes in polypeptide concentration, we have observed increased conversion of starting material, and a corresponding decrease in the a / €ratio of products (from 27 to 14), when polypeptide concentration is decreased from 600 to 60 FM in the modification of FGAKQA with one 1% (vol) addition of reagent in 100 mM N-ethylmorpholine hydrochloride, pH 6.9. The chemistry described in this paper should give yields of final product comparable to yields obtainable by de novo solid-phase synthesis of an appropriately derivatized peptide. It was designed primarily with the idea that resulting reaction mixtures could be used with no purification except elimination of excess iodoacetate, but it is also possible to purify the peptide products in the same way that peptides synthesized by organic chemistry methods are purified. Iodoacetylated peptides can be purified by HPLC and stored for a t least 1 year in aqueous solution at -20 “C without losing their capacity to alkylate sulfhydryl groups (data not shown). The reagent iodoacetic anhydride is commercially available, and stock solutions are stable when stored over drying agent at -20 “C. Potential Applications. One motivation for developing this chemistry is the increasing number of important polypeptides being made available in large amounts by recombinant DNA techniques. In addition, the method should be applicable to chemical or protease-generated fragmentation products of polypeptides; an especially attractive set of peptides are those derived from digestion with proteases with trypsin-like specificities, since the fragments will have no more than one Lys per fragment. There are many potential applications of this chemistry and the iodoacetylated peptides it can produce; among them are the following. I. Cross-Linking of a Polypeptide to a Carrier Molecule, Such as a Protein, for Use as a n Immunogen to Raise Antibodies to the Polypeptide. It is significant that for this and other applications to succeed the structure of the polypeptide need not be known, except that it must have a free N-terminus. For example one can imagine using this chemistry to make antibodies to a newly isolated peptide factor even before its structure has been assigned. It is also significant that the chemistry can be done effectively on mixtures of peptides. Thus, for example, it should be possible to test various fragmentation methods for their ability to generate highly immunogenic fragments of a protein, as might be useful in a search for a maximally effective polypeptide vaccine. 2. Cross-Linking of a Polypeptide to a Solid Support for Use as a n Affinity Column. Although the acetyl group does not contribute much to the peptide as a spacer, it is equally possible to add the spacer to the matrix as part of the cross-linking chemistry. For example, we have reacted Lys-sepharose with 2-IT to generate a thiol sepharose with a spacer composed of both the tetramethylene arm of Lys and the trimethylene arm of cleaved 2-IT (R.W., unpublished). Here too there may be special advantages to being able to immobilize peptides of
Bioconjugate Chem., Vol. 1, No. 2, 1990 121
unknown structure or mixtures of peptides. For example, one could immobilize the peptides derived from one or more proteolytic digests of a protein in order to make an immunoaffinity column specific for “linear epitope” antibodies to the protein. 3. Modify Proteins or Other Molecules in Structure/ Function Studies. As shown above, iodoacetylated peptides can be reacted with Cys residues of proteins. When the Cys is not an N- or C-terminal residue, this would lead to novel branched polypeptides. Polypeptides modified with a-haloacetyl moieties may also prove to be useful as affinity labeling reagents, in analogy with this use of other a-halocarbonyl derivatives (16). Such reagents have potential as diagnostic tools for structure/function relationships and also as drugs. 4 . Tagging of Polypeptides with Reporter Groups f o r Biophysical and Other Studies. It should be possible to label polypeptides with biotin, fluorescent probes, radioactive groups, etc., by using this chemistry. For example, radiolabeled polypeptides with reproducible properties could be prepared by synthesis of a stock of iodoacetylated polypeptide, which could be conveniently and safely synthesized and purified. When needed, small amounts could be reacted with radiolabeled groups such as [35S]sodium sulfide or cysteine, the latter of which can be obtained a t 600 Ci/mmol. Although selectivity of iodoacetic anhydride will probably be considerably lower in reactions with globular proteins, because of the likelihood of a large number of Lys residues, this method may compare favorably with other methods currently used to tag proteins. 5. Cross-Linking of One Polypeptide to Another. Crosslinking of one protein to another, for example, to tag an antibody with an enzyme as a reagent for ELISA, normally involves highly nonspecific chemistry and can result in poorly characterized, cross-linked peptide reagents with properties sometimes difficult to reproduce. Iodoacetylation may provide a more selective chemistry for preparing such reagents. N-Terminally iodoacetylated peptides can also be cross-linked to each other, for example, by reaction with a limiting amount of dithiol. One attractive possible application of this chemistry is the “random reassociation” of peptide fragments of a protein, which may be capable of generating, in a small percentage of the reaction mixture, molecules which contain a molecular surface which approximates the bioactive surface of the parent protein. Isolation and identification of such a peptide might rapidly provide leading evidence, for example, to the identity of the receptor binding site of a protein hormone. There are several reported examples of the reconstruction of antibody-binding sites of a protein by linking two discontinuous elements of the polypeptide chain in a single, relatively small peptide molecule (17, 18). Also supporting this idea is the work of Oas and Kim (19), who have recently shown that covalent association of two fragments of BPTI promotes noncovalent interactions to generate a cooperative folding unit structurally related to BPTI itself. 6. Simplify Amino Acid Sequence Analysis of Peptides from M S Fragmentation Patterns. N-Terminally iodoacetylated peptids can be used to prepare products containing a quaternary ammonium, charged group on the N-terminus; in the MS, such localizations of full positive charge generate spectra enriched in signals from fragments containing the positive charge (20). With a charge on the N-terminus derived from reaction of iodoacetylated peptides with thiocholine and other reagents, sequences can be read directly from the MS fragmenta-
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tion pattern (R.H., J.S., and R.W., manuscript in preparation). In the tandem MS, fragmentation can be directed a t any ion from a mixture of molecular species, so it may be possible to derivatize even small amounts of peptide and analyze the crude reaction mixture to obtain a sequence by this method. ACKNOWLEDGMENT
We wish to thank John Burnier (Genentech), Bill Hancock (Genentech), Richard Vandlen (Genentech), Mark Louden (Purdue), and Markus Graf (Ciba-Geigy,St. Aubin, Switzerland) for helpful suggestions and discussions and E. Rinderknecht and L. E. Burton for the murine interferon-gamma. LITERATURE CITED (1) Green, N., Alexander, H., Olson, A., Sutcliffe, J. G., and Lerner, R. A. (1982) Immunogenic structure of the influenza virus hemagglutinin. Cell 28, 477-487. (2) Greenstein, J. P., and Winitz, M. (1961) Chemistry of the Amino Acids pp 486-488, Wiley, New York. (3) Means, G. E., and Feeney, R. E. (1971) Chemical Modification of Proteins, pp 14-15, 68-71, Holden-Day, San Francisco. (4) Stark, G. R. (1965) Reactions of cyanate with functional groups of proteins. 111. Reactions with amino and carboxyl groups. Biochemistry 4 , 1030-1036. ( 5 ) Hunter, M. J., and Ludwig, M. L. (1962) The reaction of imidoesters with proteins and related small molecules. J . Am. Chem. SOC. 84, 3491-3504. (6) Anderson, W. (1956) C. R. Trau. Lab. Carlsberg, Ser. Chim. 39, 104. (7) Evans, R. L., and Saroff, H. A. (1957) A physiologicallyactive guanidinated derivative of insulin. J . Biol. Chem. 228, 295304. (8) Cole, R. D. (1961) On the transformation of insulin in concentrated solutions of urea. J . Biol. Chem. 236, 2670-2671. (9) Bradbury, J. H., Howell, J. R., Johnson, R. N., and Warren, B (1978) Introduction of a strong binding site for lanthanides a t the N-terminus of peptides and ribonuclease. Eur. J . Biochem. 84,503-511. (10) Desbuquois, B. (1975)Acetylglucagon:preparation and characterization. Eur. J. Biochem. 60, 335-347. (11) Wetzel, R., Heyneker, H. L., Goeddel, D. V., Jhurani, P., Shapiro, J., Crea, R., Low, T. L. K., McClure, J. E., Thurman, G. B., and Goldstein, A. L. (1981) Production of biologically active Ne-desacetylthymosin a1 in Escherichia coli through expression of a chemically synthesized gene. In Cellular Responses to Molecular Modulators; Miami Winter Symposium (L. W. Mozes, W. A. Scott, J. Schultz, and R. Werner, Eds.) Vol. 18 pp 251-270, Academic Press, New York. (12) Magee, A. I., Grant, D. A. W., Hermon-Taylor, J.,and Offord, R. E. (1981) Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from a- [3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity. Biochem. J . 197, 239-244. (13) Gray, P. W., and Goeddel, D. V. (1983) Cloning and expression of murine immune interferon cDNA. Proc. Natl. Acad. Sci. U.S.A. 80, 5842-5846. (14) Moore, G. J. (1985) Kinetics of acetylation-deacetylation
Wetrel et al.
of angiotensin 11. Int. J. Pep. Protein Res. 26, 469-481. (15) Riordan, J. F., and Vallee, B. L. (1967) 0-Acetyltyrosine. Methods Enzymol. 11,570-576. (16) Murdock, G. L., Warren, J. C., and Sweet, F. (1988) Human placental estradiol 17P-dehydrogenase:Evidence for inverted substrate orientation (“wrong-way”binding) a t the active site. Biochemistry 27, 4452-4458. (17) Atassi, M. Z., and Habeeb, A. F. S. A. (1977) In Zmmunochemistry of Proteins (M. 2. Atassi, Ed.), Vol. 2, pp 177264, Plenum Press, New York. (18) Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986) A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23, 709-715. (19) Oas, T. G., and Kim, P. S. (1988) A peptide model of a protein folding intermediate. Nature 336, 42-48. (20) Hopper, S., Johnson, R. S., Vath, J. E., and Biemann, K. (1989) Glutaredoxin from rabbit bone marrow: Purification, characterization, and amino acid sequence determined by tandem mass spectrometry. J. Biol. Chem. 264, 20438-20447. Registry No. GRTQRLQTLTNLF, 125685-76-9;SRVSQRGRLTLNLESGRWR, 125713-32-8; norleucine-FPTIPLSR, 125713-33-9; EVVEEAN, 69871-75-6; SDAAVDTSSEITTK, 125685-77-0; SDAAVDTSSEITTKDLKEKKEVVEEAEN, 125685-78-1; AGAKQA, 125685-79-2; RGAKQA, 125685-80-5; NGAKQA, 125685-81-6; BGAKQA, 125685-82-7; EGAKQA, 125685-84-9; QGAKQA, 125685-85-0; GGAKQA, 125685-86-1; HGAKQA, 125685-87-2; IGAKQA, 125685-88-3; LGAKQA, 125685-89-4;KGAKQA, 125685-90-7; MGAKQA, 125685-91-8; FGAKQA, 125685-92-9; PGAKQA, 125685-93-0; SGAKQA, 125713-34-0;TGAKQA, 125685-94-1; WGAKQA, 125685-95-2; YGAKQA, 125685-96-3; VGAKQA, 125685-97-4; a-iodoacetylAGAKQA, 125713-35-1;a-iodoacetyl-RGAKQA, 125713-36-2; aiodoacetyl-NGAKQA, 125713-37-3; a-iodoacetyl-BGAKQA, 125713-38-4;a-iodoacetyl-EGAKQA, 125713-40-8;a-iodoacetylQGAKQA, 125713-41-9;a-iodoacetyl-GGAKQA, 125713-42-0;aiodoacetyl-HGAKQA, 125713-43-1; a-iodoacetyl-IGAKQA, 125713-44-2;a-iodoacetyl-LGAKQA, 125713-45-3;a-iodoacetylKGAKQA, 125713-46-4; a-iodoacetyl-MGAKQA, 125713-47-5; a-iodoacetyl-FGAKQA, 125713-48-6; a-iodoacetyl-PGAKQA, 125713-49-7;a-iodoacetyl-SGAKQA, 125713-50-0;a-iodoacetylTGAKQA, 125713-51-1; a-iodoacetyl-WGAKQA, 125713-52-2; a-iodoacetyl-YGAKQA, 125713-53-3; a-iodoacetyl-VGAKQA, 125713-54-4; bis(iodoacety1)-AGAKQA, 125713-55-5; bis(iodoacety1)-RGAKQA, 125713-56-6; bis(iodoacety1)NGAKQA, 125713-57-7;bis(iodoacety1)-BGAKQA, 125713-588; bis(iodoacety1)-EGAKQA, 125713-60-2; bis(iodoacety1)QGAKQA, 125713-61-3; bis(iodoacety1)-GGAKQA, 125713-624; bis(iodoacety1)-HGAKQA, 125713-63-5; bis(iodoacety1)IGAKQA, 125713-64-6;bis(iodoacety1)-LGAKQA, 125713-65-7; bis(iodoacety1)-KGAKQA, 125713-66-8; bis(iodoacety1)MGAKQA, 125713-67-9;bis(iodoacety1)-FGAKQA, 125713-680; bis(iodoacety1)-PGAKQA, 125713-69-1; bis(iodoacety1)SGAKQA, 125713-70-4; bis(iodoacety1)-TGAKQA, 125713-715; bis(iodoacety1)-WGAKQA, 125713-72-6; bis(iodoacety1)YGAKQA, 125713-73-7; bis(iodoacety1)-VGAKQA, 125713-748; AlaGly, 687-69-4;AspGly, 3790-51-0; GluAla, 21064-18-6; GlyGly, 556-50-3; HisGly, 2578-58-7; IleAsn, 59652-59-4; LeuGly, 686-50-0; MetGly, 14486-03-4;PheGly, 721-90-4; ProGly, 257857-6; SerGly, 687-63-8; TrpGly, 7360-09-0; TyrGly, 673-08-5;ValGly, 686-43-1; AcGlyLysOCH,, 10236-44-9;CYSSSCHZCH~OH, 125685-83-8; a-iodoacetyl-CysSSCH,CH,OH, 125713-39-5; bis(iodoacety1)-CysSSCH,CH,OH, 125713-59-9;iodoacetic anhydride, 54907-61-8; 2-iminothiolane, 6539-14-6.
