Identification of Cysteine As the Reactive Group in Pyruvate Kinase Alkylated by 5-Chloro4.Oxopentanoic Acid by R

Biochem. J. (1976) 159,213-219 213 Printed in Great Britain

Identification of Cysteine as the Reactive Group in Pyruvate Kinase Alkylated by 5-Chloro4.oxopentanoic Acid By R. ALAN CHALKLEY and DAVID P. BLOXHAM Department ofPhysiology and Biochemistry, University of Southampton, Southampton S09 3TU, U.K. (Received 8 March 1976)

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro4oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro- 4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.

In the preceding paper (Bloxham & Chalkley, (Bloxham & Chalkley, 1976). In all these experiments 1976), 5-chloro-4-oxopentanoic acid was shown to the reaction was terminated by adjusting the pH to specifically alkylate rabbit muscle pyruvate kinase at 7.0 with 1 M-acetic acid before 70% loss of activity to a group which might be located close to the 'phos- eliminate any problems due to protein aggregation. phoryl overlap' region of the active site (Reynard et For the experiments involving amino acid analysis or al., 1961). The next stage in the study of this reaction periodate oxidation the protein was reduced with was to identify the nature of the functional group at 5mg of NaBH4. All the 5-chloro-4-oxopentanoate- the active site. The report describes the synthesis of a labelled pyruvate kinase samples were exhaustively standard reference amino acid (4-hydroxypentanoic dialysed against water at 4°C and finally freeze-dried. acid alanine thioether, I) and the use of performic acid oxidation to analyse the labelled amino acid. Degradation by performic acid oxidation should be Chromatographic analysis ofmodified amino acids particularly useful in this system, since the proposed structure of the inhibitor amino acid adduct Labelled pyruvate kinase (25mg) was hydrolysed in at 0 lOml of6M-HCI 110°C for 18h. The hydrolysate 11 was flash-evaporated and then vacuum-dried twice ml water. was (X-CH2--C-CH2--CH2--CO2H) means that the from 5 of High-voltage electrophoresis compound should be split into two chemically performed with either a Gilson high-voltage electro- identifiable intermediates (see below for detailed phorator model D or Locarte electrophoresis explanation). Although performic acid oxidation has apparatus. The buffers used were pH 1.9 (acetic acid/ been used previously to produce carboxymethyl- formic acid/water; 27:25:888, by vol.), pH3.5 amino acids from more complex amino acids (Petra (pyridine/acetic acid/water; 1:10:289, byvol.), pH4.7 et al., 1965; Husain & Lowe, 1968), the structure of (pyridine/acetic acid/water; 1:1:48, by vol.), and the degraded derivative allowed only the carboxy- pH6.5 (pyridine/aceticacid/water; 25:1:475, byvol.). methyl-amino acid to be analysed, and this meant Amino acids were located as either ninhydrin- that complete information on the mechanism of this positive spots or by radioactivity. For initial location reaction for protein-bound intermediates was not of radioactive spots a Camlab radiochromatogram obtained. The present communication shows that scanner was used. Subsequently the paper was cut there is a high degree of preference in the cleavage into 1 cm strips, soaked in 1 ml of water for 1 h and reaction. Finally, it is suggested that periodate then counted for radioactivity. Amino acid analyses oxidation may be advantageous in the preliminary of hydrolysed samples were performed with a JEOL screening to identify the amino acid. JLC-6AH amino acid analyser. The column for acidic and neutral amino acids contained JEOL Materials and Methods LCR2 resin. For radioactive samples, a stream splitter was used before the ninhydrin-analysing Pyruvate kinase modification system and approx. 10% of the eluate from the The exact conditions for the bulk modification of column was collected in a fraction collector. The pyruvate kinase were described in the previous paper entire sample was counted for radioactivity. Vol. 159 214 R. A. CHALKLEY AND D. P. BLOXHAM

Performic acid oxidation vacuo. After acid hydrolysis, the amino acid analyser Performic acid was prepared by the addition of showed the presence of glutamate, glycine and a new H202 (30%, v/v) to formic acid (firs, 1956). In a amino acid, which was presumably compound I typical experiment, 25mg of 5-chloro-4-oxo[3,5-3H]- (Fig. 1). To confirm this, we purified the amino acid pentanoate-labelled material was dissolved in 0.5ml and identified its structure by n.m.r. spectroscopy. of formic acid, cooled on ice for 30min, andthen 1 ml Fortunately, the mixture of amino acids was easily of performic acid solution was added. After 2.5h at purified by high-voltage electrophoresis at pH3.5. 0WC, the sample was diluted with 25ml of water and After performing this chromatographic purification freeze-dried. The sample was hydrolysed and ana- twice, the modified amino acid gave only a single lysed by electrophoresis. N.-3-Carboxymethylhisti- ninhydrin-positive and radioactive peak in the dine and e-carboxymethyl-lysine (Gundlach et al., amino acid analyser. The n.m.r. spectrum of this 1959) were synthesized by Dr. G. Rasool of this compound was recorded in 2H20 by using a Varian department. XL100-12 machine operated in the CW mode at 100MHz with a "IF lock and extemal trimethylsilyl reference, The hydrogen resonances observed were as Periodate oxidation follows; 3 1.8-2.8p.p.m., 4H, multiplet, -CH2 The NaBH4-reduced 5-chloro4-oxo[3,5-3H]pen- CH2-; a 2.85p.p.m., doublet, S-CH2C; tanoate-labelled samples were oxidized with 100mg 2H, of NaIO4 in lOml of satd. NaHCO3 containing 02H 10mg ofcarrier formaldehyde. After 16h the niixture 3 3.05p.p.m., 2H, triplet, C-CH2-S; a 4.6-4.9 was adjusted to pH6 with 50% (w/v) acetic acid, and I formaldehyde was isolated as the dimedone complex N2H2 (Zaman et al., 1973). p.p.m., 1H, multiplet, --H. This spectrum ac- 5.Amino.4hydroxy[5-3H2]pentanoic acid was I donated by Dr. P. M. Jordan of this department. N2H2 counted for all the expected resonances. The methyl- Results and Discussion ene group adjacent to the amino group Synthesis and characterization of4-hydroxy[3,5_3H]- H-CH2-S in compound I is not a simple pentanoic acidalanine thioether (I) The first task in identifying the amino acid in \N2H2 pyruvate kinase modified by 5-chloro-4-oxopentanoate was to synthesize an authentic reference amino acid. For this purpose we chose 4-hydroxypentanoic 1.0o In acid alanine thioether (I), since thealkylation reaction 0 was easily monitored by 5,5'-dithiobis-(2-nitro- V 0.8 8 benzoate) titration (Ellman, 1959). Further, gluta- 0in v: thione presents a useful N-blocked cysteine deriva- o 0.6 6 *5 tive, since its adducts are readily precipitated as the ._ barium salt, which aids purification. U U .* 0.4 4 .... OH NH2 .0 . 04 I ..r- 0.2 2 ,,x CO2H-[CH2]2-CH-CH2-S-CH2--CH-CO2H z o" (I) 120 140 160 180 200 220 240 260 Glutathione (0.3mmol) was allowed to react with Retention time (min) 5-chloro4oxo[3,5-3Hlpentanoate (0.42mmol; 2.5 x Fig. 1. Chromatography of 4-hydroxy(3,5-3-H]pentanoic 103d.p,m./nmol) in water, pH8 (maintained with acidalanine by using the amino acidanalyser IM-NaOH) for 2h at room temperature (20°3). About 80 % ofthereactionwascompletewithin 0min This Figure shows the chromatography of 4-hydroxy- as judged by both 5,5'-dithiobis-(2-nitrobenzoate) [3,5-3H]pentanoic acid alanine thioether, which was titration and alkali uptake. The carbonyl group in the produced either synthetically from glutathione or by alkylation of pyruvate kinase. The trace shows the elution adduct was then reduced with NaBH4 and after profile from only the acidic and neutral column of the 30min an excess of barium acetate (4m1, 1M) was amino acid analyser. o, Ninhydrin-positive trace whlen added. The barium salt was precipitated with 4vol. 5- chloro-4-oxo[3,5-3H]pentanoate-labelled glutathione of ice-cold ethanol and collected by centrifugation, was analysed. *, Distribution of radioactivity when After washing three times with ice-cold ethanol and 5-cWoro-4'oxo[3,5_3H]pentanoate-labelled pyruvate kin- twice with diethyl ether the product was dried in ase was analysed. 1976 ALKYLATION OF CYSTEINE BY 5-CHLORO-4-OXOPENTANOIC ACID 215 doublet, but is more complex. This may be due to the derived from position 3 of5-chloro-4-oxopentanoate) fact that it is adjacent to an asymmetric centre which and [3H]formaldehyde. This route gives no informa- was derived from the L-cysteine in glutathione. This tion on the nature of the amino acid modified. In may be contrasted with the methylene group adjacent contrast, route (b) leads to the formation of the [3H]- to the hydroxyl group (S-CH2-CH- which is a carboxymethyl-amino acid (eH derived from position 5 of 5-chloro-4-oxopentanoate), which can be k 02H1 identified by comparison with standards. [3H]- simple doublet, reflecting that the NaBH4 reduction Hydroxypropionate formed by route (b) will be of the carbonyl produces a racemic mixture. volatile and not analysed. It follows that analysis of Having satisfactorily demonstrated that the syn- the distribution of isotope between succinate and thetic amino acid corresponds to 4-hydroxypentanoic the carboxymethyl-amino acid will enable both the alanine thioether, it was compared with the modified identification of the labelled amino acid and also the amino acid derived from 5-chloro-4-oxopentanoate- migration preferred in the reaction mechanism. labelled pyruvate kinase. Fig. 1 shows that after Performic acid oxidation was performed on 5-chloro- acid hydrolysis of labelled pyruvate kinase, a single 4-oxo[3,5-3H]pentanoate, the 5-chloro-4-oxo- radioactive peak was obtained which corresponds [3,5-3H]pentanoate glutathione adduct and 5-chloro- exactly to 4-hydroxypentanoic acid alanine thioether. 4-oxo[3,5-3H]pentanoate-labelled pyruvate kinase. The identity of the modified amino acid was con- The first two samples served as controls. Under our firmed by high-voltage electrophoresis, which showed experimental conditions, there was only a 5-6 % that in four solvents the synthetic amino acid and the recovery of radioactivity as involatile material after modified amino acid derived from pyruvate kinase performic acid oxidation and acid hydrolysis. Loss of had identical chromatographic properties. When radioactivity was presumably due to both acid- electrophoresis was carried out at 5kV and l5mA for catalysed detritiation of the ketone before oxidation 2h, the observed mobilities towards the cathode were and loss of volatile products. The involatile products as follows; pH 1.9, 7.5 ± 0.5cm; pH3.5, 5.5 ± 0.5 cm; were analysed by electrophoresis in pH3.5 buffer at pH4.7, 3.2±0.4; pH6.5, 1.4± 0.4. Both the synthetic 3.5kV and 50mA for 45min. Under these conditions amino acid and the derivative from pyruvate kinase the mobilities of standards were: carboxymethyl- failed to move towards the anode at pH values lysine, -3.4 ± 0.5cm; N-3-carboxymethylhistidine, greater than 3.5 (approximate pK, of CO2H). This -2.0 ± 0.4cm; succinate, +1.50 ± 0.4cm; carboxy- suggests that compound I may be isolated as the methylcysteine, +6.5 ± 0.5cm. The following obser- lactone with one less negative charge than expected. vations were made on the degradation procedure. Note that carboxymethylcysteine does migrate (1) 5-Chloro-4-oxo[3,5-3H]pentanoate (1.5 x 104 towards the anode at pH3.5. d.p.m.) only gave rise to [3H]succinate (l.5 x 104 d.p.m.), and there were no radioactive peaks in the Performic acid oxidation of the 5-chloro-4-oxo- vicinity of any of the standard carboxymethyl-amino pentanoate-modified amino acid in pyruvate kinase acids. (2) Both 5-chloro-4-oxo[3,5-3H]pentanoate glutathione adduct and the 5-chloro-4-oxo[3,5-3H] Although the initial experiment gives strong pentanoate-modified amino acid from pyruvate evidence in favour ofcysteine as the reactive group on kinase gave rise to [3H]carboxymethylcysteine as the pyruvate kinase, the result is ambiguous because the sole radioactive carboxymethylamino acid. This lysyl 5-chloro-4-oxopentanoate adduct was not confirms unambiguously that the amino acid modi- prepared. Attempts to prepare an authentic sample fied by this compound is cysteine and eliminates both of this compound by using either polylysine or t-N- histidine and lysine. (3) When the distribution of acetyl-lysine failed to give the authentic compound. radioactivity between succinate and carboxymethyl- We chose to use performic acid oxidation (Baeyer- cysteine was compared, 5-chloro-4.oxo[3,5-3H]- Villiger reaction; House, 1972) to degrade the pentanoate glutathione gave 1.3 x 104d.p.m. in modified amino acid. The rationale behind this [3H]succinate and 2 x 103d.p.m. in [3H]carboxy- experiment is illustrated in Scheme 1. methylcysteine. Similarly, the 5-chloro4-oxo- The reaction involves treatment with performic [3,5-3H]pentanoate-modified amino acid from pyru- acid, and, as illustrated, while the initial structure is vate kinase gave 1 x 104d.p.m. in [3H]succinate and intact, acid-catalysed enolization causes loss of 3H to 5 x 103d.p.m. in [3Hjcarboxymethylcysteine. The the medium so that a low final recovery of 3H is formation of substantially higher amounts of [3H]- anticipated. Performic acid causes cleavage of either succinate shows that reaction pathway a (Scheme 1) C-C bond adjacent to the carbonyl. Usually the occurs preferentially. The preference is of the order oxidation shows a distinct preference in the cleavage of 70-80%, which is similar to the specificity of reaction according to which alkyl group migrates performic acid oxidation in other reactions (House, most readily (House, 1972). If route (a) is followed 1972). This result clearly demonstrates that the then the eventual products will be [$Hisuccinate (3H successful application of performic acid degradation Vol. 159 216 R. A. CHALKLEY AND D. P. BLOXHAM

CH2CO2H. CH2CO2H CH2COZH I cmCW2 H+-catalysed CIV2* .H+-catalysed CH* enolization I 11' C-OHII enolization C-OH 11 IH CH* CHm ' cX -*H+ XProtein XProtein - - XProtei.n

H | HC NHNOH Oe -* Cl' * Protein-X-CH2--C-CH2-CH2-CO2H al I4)b

a 1 (~~~~N /HC

ProteinX CH2-0-C--CH2-CH2-CO2H ProteirH-XCH2-C-0-CH2-H202

*o * ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~411H+ Amino acid-X-CH20H + C02H-CH2-CH2-CO2H Amino acid-X-CH2-C02H + HO-CH2-CH2-CO2H

Amino acid-X- + CH20 i Involatile products analysed Volatile Volatile Scheme 1. Performic aciddegradation of5-chloro4oxo[3,5-3H]pentanoate-modifiedpyruvate kinase The scheme shows the general pathway for performic acid degradation of a 5-chloro-4-oxo[3-5-3H]pentanoate-labelled amino acid. X may be Cys, Lys or His. The asterisk indicates the presence of 3H.

depends on the exact nature of the alkylating group 5-chloro4-oxopentanoate is a cysteine thiol. The for the protein and its influence on migration prefer- methods used for establishing this reaction are ence (pathway a versus b, Scheme 1). A high migra- rigorous, but they are also time-consuming and quite tion preference of this type might explain why complicated. One can readily imagine circumstances performic acid oxidation of 1,3-dibromoacetone- where the structure ofthehalogenomethyl ketonemay linked histidine and cysteine in papain gave rise to be sufficiently complex to preclude the synthesis of only carboxymethylhistidine (Husain & Lowe, 1968) the inhibitor amino acid adduct. Indeed, it is probable rather than an equal mixture of both carboxymethyl- that the authentic lysyl adduct cannot be made bythe cysteine and carboxymethylhistidine. simple reaction between the amine and halogeno- 0 Periodate oxidation: a simple methodfor distinguish- 11 ing between thiol and amino alkylation by halogenomethyl ketone (R1-NH2+X CH2-C-R2 methyl ketone compounds 0 The previous experiments have proved unambigu- 11 ously that the group modified in pyruvate kinase by HX+R1-NH-CH2---C-R2) because this reaction 1976 ALKYLATION OF CYSTEINE BY 5-CHLORO4OXOPENTANOIC ACID 217

0 N, 11 NaBH4 CH-OH NaIO4 R'-C,-CH2-SR I * No reaction CH2-S :R-SH / / C R (aI) 0 11 * RW-C-CH2CI

d R-NHa 0 RI 11 * NaBH4 CH--OH NaIO4 Rl-C-H2-NHR ) -* R'CHO CH2-NH R-N RNH2 11 R CHW CH2O (III) Scheme 2. NaBH4 reductionfollowed byperiodate oxidation as a generalmethodfor identifying thefunctionalgroup modified by a halogenomethyl ketone alkylating reagent The scheme shows the reaction ofa general chloromethyl ketone with either a protein-bound thiol (R-SH, route c) or amino (R-NH2, route d). For 5-chloro-4-oxo[3,5-3H]pentanoate, R1 is C02-CH2-CH2. The asterisk indicates an isotopic label which could be either 14C or 3H. Note that for 5-chloro-4-oxo[3,5-3H]pentanoate if the reaction proceeds through route d, then only 3H at C-S (50%/) will be liberated by this procedure. Normally, the labelled protein would be hydrolysed before the periodate oxidation. Analysis is finally achieved by measuring the isotope liberated as formaldehyde.

proceeds preferentially through the Schiff base (D. failure of compounds of structure type II to give Evans, I. Climie & M. Akhtar, personal communi- labelled formaldehyde is shown by Expts. 1-4 (Table cation). Further, as we have demonstrated quite 1) where both synthetic cysteine 5-chloro-4-oxo- clearly, the success ofthe application ofthe performic pentanoate adducts and pyruvate kinase 5-chloro4- acid degradation depends on the good fortune with oxopentanoate adducts were tested. The observation regard to the rate of proton exchange adjacent to the that the pyruvate kinase 5-chloro-4-oxopentanoate carbonyl group and the preference obtained in the adduct gave no [3H]formaldehyde is consistent with migration reaction. cysteine as the group in pyruvate kinase alkylated by Since halogenomethyl ketone modifications of 5-chloro-4-oxopentanoate. The first four experiments proteins will continue to be a common procedure in quoted in Table 1 givenegative results and wouldonly enzyme chemistry, we felt that it might be appropriate be verified by successful degradation of a compound to point out a convenient method that allows some of structure type (III). We have been unable to syn- distinction between whether thiol or amino groups thesize the authentic 5-chloro-4-oxopentanoate lysine are reacting with the halogenomethyl ketone group. adduct, but we had in our laboratory a sample of a The method simply involves NaBH4 reduction related analogue 5-amino-4-hydroxy[5-3H]pentanoic followed by periodate oxidation (Scheme 2). The acid. This was obtained from biosynthetic experi- method is based on the fact that periodate oxidation ments with 5-aminolaevulinate synthetase to con- will occur only if a cyclic ester is formed as follows vert [2-3H2]glycine and succinyl-CoA (carboxy- (Bunton, 1965) propionyl-CoA) into 5-amino-4-oxo[5-3H]pentanoic acid, which was reduced with NaBH4 (Zaman et al., 1973). It is apparent that, as expected, this type M C-OH NaIO4 C-Ox /0- structure gives a high yield of 13H]formaldehyde I| I-OH C-XH (Expt. 5, Table 1). As final proofofthe validity ofthis I 0 method, Meloche (1973) has shown that periodate oxidation ofcompound IV leads to the production of where X = 0,5S, NH or N-alkyl. [3H]formaldehyde in excellent yield (shown as Expt. From Scheme 2 it is clear that intermediate Ill has 6, Table 1 for convenience). We feel that these the correct configuration for periodate oxidation, experiments demonstrate that NaBH4 reduction of whereas intermediate II should not react. The the amino acid radioactive inhibitor adduct followed Vol. 159 218 R. A. CHALKLEY AND D. P. BLOXHAM

Table 1. Periodate degradation oflabelledamino acids The details of the periodate oxidation and isolation of formaldehyde as the dimedone are described in the Materials and Methods section. For Expts. 1-4 the samples were reduced with NaBH4 before oxidation of the intermediate used in the periodate oxidation. The percentage recovery in formaldehyde in these experiments was corrected for the fact that only 50% of 3H-labelled 5-chloro-4-oxopentanoate (COP) is at position 5 which gives rise to formaldehyde. The data in Expt. 6 are taken from Meloche (1973). 3H in Expt. Total 3H formaldehyde % recovery in no. Sample degraded (d.p.m.) (d.p.m.) formaldehyde 1 [3,5-3H]COP glutathione adduct 2.6x 106 3.7x 104 2.85 2 4-Hydroxy[3,5-3H]pentanoic acid t.5 x 105 1.5x 103 2.0 alanine thioether 3 [3,5-3H]COP pyruvate kinase adduct 1.22 x 105 2x103 3.27 (tryptic digestion) 4 [3,5-3H]COP pyruvate kinase adduct 3.11 x 105 1.8 x 103 1.15 (total acid hydrolysis) 5 5-Amino-4-hydroxy[5-3H]pentanoic acid 5x104 4.7 x 104 94 6 Compound IV 5.5 x 103 5x103 90.0 CH20H /CH-NH C02H [CH2J3 CHNH2 CO2H by periodate oxidation and analysis of [3H]form- analysed by two-dimensional chromatography. The aldehyde production provides a most useful initial sample was subjected to descending chromatography procedure in the identification of the labelled amino in butan-1-ol/acetic acid/water (4:1:5, by vol., upper acid in a protein. As a note of caution it must be phase) for 21 h followed by electrophoresis in pointed out that this procedure does not distinguish pyridine/acetic acid/water (1:10:280, by vol.) at between alkylation reactions with either cysteine or 2000V and 120mA for 75min. After ninhydrin imidazole. Therefore this procedure will probably staining 56 peptides were located out of a predicted have its maximum utility, provided there are good 66 based on the number of lysine and arginine reasons to expect that either a cysteine or lysine has residues (Cottam et al., 1969). Each peptide was cut been modified. out, soaked in 0.5ml ofwater for 1 h and analysed for radioactivity. Out of all the peptides, three contained Preliminary analysis of 5-chloro-4-oxopentanoate- radioactivity ofwhich the majority (74 %) was located labelledpeptides in a peptide region that migrated 6.5cm and 13.5cm by chromatography and electrophoresis respectively. 5-Chloro-4-oxo[3,5-3H]pentanoate-labelled pyru- This result substantiates that the site of action of vate kinase (80mg; 15x106d.p.m.; 1.2mol of 5-chloro-4-oxopentanoate appears to be directed inhibitor per mol ofprotomer) was dissolved in Omil selectively towards one region ofthe protein. of SOmM-Tris/HCI, pH8.2, containing 6M-urea. An initial attempt has been made to purify this First, the carbonyl group was stabilized by peptide further. After tryptic digestion, peptides were reduction with NaBH4 (5mg; 1 h). The protein was resolved from undigested material by chromato- reduced (80mg of dithiothreitol; 30°C; 3h), and graphy on a column (100cmx2cm) of Sephadex carboxymethylated (iodoacetic acid, 400mg; G-10. The peptides were then fractionated on a NaHCO3, 200mg; 4h; 30°C). The protein was ex- column (17cm x 2.5cm) ofDEAE-cellulose by using a haustively dialysed against 6 x 5 litres of water (72h 500ml gradient from 25mM- to 1M-NH4HCO3. The at 4°C). Virtually all the radioactivity was retained. majorradioactive peak (80 %)waselutedwith 100mM- Tryptic digestion was perfonned for 5h at 370C in NH4HCO3. This was further purified by descending 16ml of 5OmM-NH4HCO3 containing 2mg of chromatography for 24h in butan-1-ol/acetic acid/ diphenylcarbamoyl chloride-treated trypsin. At the water (4:1:5, by vol., upper phase). Of the eight end of digestion, trypsin was denatured at 90°C and peptides located by ninhydrin only one peptide con- removed by centrifugation. A suitable sample (5mg tained all of the radioactivity (mobility 7cm). The of protein) was freeze-ried, dissolved in water and overall yield of this peptide was only 3 % which was 1976 ALKYLATION OF CYSTEINE BY 5-CHLORO-4-OXOPENTANOIC ACID 219 inadequate for sequence work with the facilities Bunton, C. A. (1965) in Oxidation in Organic Chemistry, available in this laboratory. The amino acid compo- Part A (Wiberg, K. G., ed.), pp. 367-407, Academic sition was shown to be Arg (1.00), Asp (1.13), Ser Press, New York and London (0.71), Glu (0.61) and Cottam, G. L., Hollenberg, P. F. & Coon, M. J. (1969) 4-hydroxy[3,5-3H]pentanoic J. Biol. Chem. 244, 1481-1489 acid-alanine thioether, (0.58). This indicates that the Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 peptide produced by the trypsin treatment is only a Gundlach, H. G., Stein, W. H. & Moore, S. (1959) J. Biol. small fragment. Chem. 234, 1754-1760 Hirs, C. H. W. (1956) J. Biol. Chem. 219, 611-621 We are grateful to Professor M. Akhtar for his in- House, H. 0. (1972) in Modern Synthetic Reactions, valuable advice on this project. R. A. C. acknowledges the pp. 321-330, W. A. Benjamin, Menlo Park, CA award ofan M.R.C. studentship. Mr. I. Climie performed Husain, S. S. & Lowe, G. (1968) Biochem. J. 108, 855-859 the n.m.r. analysis. Meloche, H. P. (1973) J. Biol. Chem. 248, 6945-6951 Petra, H. P., Cohen, W. & Shaw, E. (1965) Biochem. Biophys. Res. Commun. 21, 612-618 Reynard, A. M., Hass, L. F., Jacobsen, D. D. & Boyer, References P. D. (1961) J. Biol. Chem. 236, 2277-2283 Bloxham, D. P. & Chalkley, R. A. (1976) Biochem. J. 159, Zaman, Z., Jordan, P. M. & Ahktar, M. (1973) Biochem. J. 201-211 135,257-263

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