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TABLE I I I (Continued) J. E. Folk and M. Gross, J. Biol. Chem. 246, 6683 (1971). b N. Richman and J. W. Bodley, J. Biol. Chem. 248, 381 (1973). J. Sedlacek, J. Jonak, and I. Rychlik, Biochem. Biophys. Acta 254, 478 (1971). d R. Lee and W. D. McElroy, Biochemistry 8, 130 (1969). • S. M. D'Ambrosio and G. I. Glover, Arch. Biochem. Biophys. 167, 754 (1975). M. J. Owen and H. P. Voorheis, Eur. J. Biochem. 62, 619 (1976). P. Rainey, E. Holler, and M. Kula, Eur. J. Biochem. 63, 419 (1976). h E. Khedouri, P. M. Anderson, and A. Meister, Biochemistry 5, 3552 (1966). i L. Frolova, G. K. Kovaleva, M. B. Agalarova, and L. L. Kisselev, FEBS Left. 34, 213 (1973). J. Silver and R. A. Laursen, Biochim. Biophys. Acta 340, 77 (1974). k I. L. Norton, M. H. Welch, and F. C. Hartman, J. Biol. Chem. 250, 8062 (1975). z F. C. Hartman, this volume [10].

modified residue as carboxymethyl cysteine following oxidation with hydrogen peroxide; however, this approach may be fraught with danger. They reference the oxidation procedure used by Woenckhaus et al. 66 that indicated carboxymethyl histidine was formed from yeast alcohol dehydrogenase that was modified by pl_ [3-(3-bromoacetylpyridinium)propyl] P~-5'-adenos-5'-yl diphosphate. A subsequent paper 87 stated that the material previously identified as carboxymethyl histidine was in fact a decomposition product as a result of harsh conditions and therefore in actuality the only modified residue was a cysteinyl. eeC. Woenckhaus, M. Zoltobrocki, J. Bergh~iuser, and R. Jeck, Hoppe-Seyler's Z. Physiol. Chem. 354, 60 (1973). 6~H. JSrnvall, C. Woenckhaus, E. Schiittle, and R. Jeck, FEBS Lett. 54, 297 (1975).

Other Examples

Additional successful applications of haloketones as affinity labels are listed in Table III.

[11] H a l o a c e t y l D e r i v a t i v e s By MEIR WILCHEK and DAVID GIVOL

A variety of haloacetyl reagents are being used for affinity labeling 1 although bromoacetyl is the predominant functional group. 2 The advantages of bromoacetyl reagents stem from the following features: 1E. Shaw, Physiol. Rev. 50, 244 (1970). 2F. Naider, J. M. Becker, and M. Wilchek, Isr. J. Chem. 12, 441 (1974).

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1. Spectrum o] reactivity. All protein nucleophiles can be alkylated by this type of reagent (Table I). Evidence for affinity labeling of cysteine,3,4 histidine,5 lysine6-s and a-amino groups, 8 tyrosine, 6,7 methionine °,16 serine, 11 and glutamic acid TM has been reported, and it is likely that aspartic acid and threonine can also be labeled. Such a wide spectrum of reactivity is important for successful labeling of residues at the reactive site. 2. Relatively low reactivity. Although cysteine can be readily labeled by haloacetyl reagents, most other residues are not being labeled significantly. Therefore a very low background and high specificity of labeling are present in affinity labeling of proteins by this type of reagent, whereas the predominant label is dictated by the structural requirement and concentration of the reagent at the site. This is illustrated by the difference in inactivation of various enzymes by haloacetyl reagents and their corresponding affinity label (Table II). The low reactivity of the bromoacetyl derivatives is also sustained toward their reaction with the solvent. Thus, these reagents decompose slowly and are stable for long reaction periods; the low reactivity secures the presence of the reagent for many hours. 3. Ease o] preparation. Bromoacetyl groups can be attached to amino groups (unstable compounds can also be prepared with compounds containing hydroxyl, sulfhydryl, and imidazole) by convenient procedures using bromoacetyl bromide, ~s bromoacetic anhydride, ~4 or bromoacetylN-hydroxysuccinimide ester3,7; synthesis with bromoacetyl-N-hydroxy-

'A. Light, Proe. Natl. Aead. Sci. U.S.A. 52, 1276 (1964). N. Sonenberg, M. Wilchek, and A. Zamir, Proe. Natl. Acad. Sei. U.SM. 70, 1423 (1973). See also this volume [73]. ' S. L. Bradbury, Y. Biol. Chem. 244, 2002 (1969). *P. Cuatreeasas, M. Wilchek, and C. B. Anfinsen, J. Biol. Chem. 244, 4316 (1969). See also this volume [38]. 7y. Weinstein, M. Wilchek, and D. Givol, Bioehem. Biopkys. Res. Commun. 35, 694 (1969). "S.-¥. Cheng, M. Wilchek, M. J. Cahnman, and J. R•bbins, Biochemistry, in press. See also this volume [48]. *F. Naider, Z. Bohak, and 3. Yariv, Biochemistry 11, 3202 (1972). See also this volume [43]. ~oW. B. Lawsonand M. J. Schramm,Biochemistry 4, 377 (1965). ~IW. B. Lawson, M. D. Leafer, A. Tewes, and G. J. S. Rao, lloppe-Seyler's Z. Physiol. Chem. 349, 251 (1968). ~*G. M. ttass and H. Neurath, Biochemistry 10, 3535 (1971). 1.p.H. Strausbauch, Y. Weinstein, M. Wilchek, S. Shaltiel, and D. Givol, Biochemistry 10, 4342 (1971). ~4D. Givol, P. H. Strausbauch, E. Hurwitz, M. Wilchek, J. Haimovich, and H. N. Eisen, Biochemistry 1O, 3461 (1971).

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TABLE I MODIFICATION OF AMINO ACID RESIDUES BY HALOACYL DERIVATIVES Protein ~

Papain ~b

Carboxypeptidase"

Amino acid modified

Cysteine

Glutamic

Product b --NH--CH--CO-I CH2 l S - - C H f - - COO--NH--CH--CO-I

(OH,), i

CO --O-- CH~-- COO--NH--CH--CO-i CH:

Carbonic anhydrase 6

I-I/stidlne N

Staphylococcal nuclease 6 MOPC--315,~

Lysine

B-Galactosidase 9 chymotrypsin TM

Methionine

N--CHf--COO-

--NH--CH--CO-t (CH: h

NH-- CHf-- CO0--NH--CH--CO-i

(CH2)2

i

H3C-- S - - C H i - - COOTrypsin 11

Serine

--NH--CH--CO-i CH2

O--CHr-- COO-

--NH--CH--CO-t

CH2 Staphylococcal nuclease 6 M O P C - - 3 1 5 is

Tyrosine

O - - C H ~ - COOa The superscript numbers refer to text footnotes. b Hydrolysis of alkylated protein gave the carboxymethylated derivative of the modified amino acid residue except for modified glutamic acid, which was reconverted to the starting amino acid.

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GENERAL METHODOLOGY

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TABLE II COMPARISON OF THE EFFECT OF AFFINITY LABELS VERSUS NONAFFINITY LABELS IN THE IRREVERSIBLE INACTIVATION OF ENZYMES

Enzyme activity (half-life) Inactivating agent

Trypsin

Chymotrypsin

Carboxypeptidase

t~-Galactosidase

Haloacyl acid or acetamide Bromoacetyl affinity label

24 hr 3 hr

>2 wk 75 rain

>3 days 25 min

24 hr 30 min

suecinimide is described here. The preparation of highly purified reagents proceeds with high yield. This facile preparation is very important when radioactive reagents are required, and radioactive labeled reagents can be prepared from radioactive bromoacetic acid. Recommended procedure in this case will be to use succinimide ester of bromoacetic acid since this avoids waste of expensive radioactive reagent. An additional advantage of using bromoaeetyl is that the introduction of the radioactive functional group is always the last step in the preparation of the reagent. 4. Preparation o] a homologous series o] reagents. Since the ligand can be prepared with side chains of different length, terminating in an amino group, affinity labeling reagents of systematically increasing size can be prepared and used as a ruler for measuring distances at the combining site2 s 5. Convenient procedures ]or identification o1 labeled residues. Since the bromoacetyl moiety is attached by an amide bond to the ligand, acid hydrolysis of the protein will always result in a carboxymethyl derivative of the labeled amino acids (Table I). Carboxymethyl amino acids can be readily analyzed with an amino acid analyzer, since their position is known, 15 or by high voltage paper electrophoresis, where the additional charge will change the mobility of the labeled amino acid. la The synthesis of earboxymethyl derivatives of amino acids as markers is achieved by alkylation with haloac~tate2 6 In some cases, the carboxymethyl derivatives are not stable arid caution is necessary in their identification. With methionine, for example, acid hydrolysis will produce carboxymethyl homocysteine and homoserine as degradation products of methionine carboxymethyl sulfonium salt~5; treatment with sulfhydryl reagents can also remove label from the modified methionine2 ~ In the F. R. N. Gurd, this series Vol. 11, p. 532 (1967). 1~H. J. Goren, I). M. Glick, and E. A. Barnard, Arch. Biochem. Biophys. 126, 607 (19t~).

1, F. Naider and Z. Bohak, Biochemistry 11, 3208 (1072).

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157

case of glutamic or aspartic acids, the ester bond formed between the amino acid and the reagent is unstable in acid and alkali, and identification should proceed in other ways, e.g., by isolation of labeled peptides.

Synthesis of Reagents

Bromoacetyl-N-hydroxysuccinimide Ester. To a solution of bromoacetic acid (139 rag, 1.0 mmole) and N-hydroxysuccinimide (115 mg, 10 mmoles) dissolved in 5 ml of dioxane is added dicyclohexylcarbodiimide (206 mg, 1.0 mmole) in 2 ml of dioxane. After 1 hr at room temperature, the dicyclohexylurea is removed by filtration and washed with dioxane. The filtrate is evaporated to dryness and crystallized from isopropanol, yield, 75%; m.p., 117% Iodoacetyl-N-hydroxysuccinimde Ester. To a solution of reerystallized iodoacetie acid 18 (185 mg, 1.0 mmole) and N-hydroxysuccinimide (140 mg, 1.2 mmoles), dissolved in 5 ml of dioxane, is added dicyclohexylcarbodiimide (250 mg, 1.2 mmoles) in 2 ml of dioxane. After I hr at room temperature the dicyclohexylurea is removed by filtration and washed with dioxane. The filtrate is evaporated to dryness and crystallized from isopropanol. Yield, 70% ; m.p., 148 °. Synthesis of Radioactive Labeled Reagents. The hydroxysuccinimide ester of radioactive bromoacetic and iodoacetic acid is prepared by a scaled-down modification of the procedure for the preparation of the corresponding nonradioactive reagent. Quantities of 14C or 3H reagents were prepared, varying from 0.1 ~mole up to 100 ~moles. When the amount used is very small, one proceeds with the next step without isolation of the product or filtration of the dicyclohexylurea. The affinity labeling reagent prepared by this method is usually purified by thin-layer chromatography. Examples appear in this volume. TM Radioactive Bromoacetic Anhydride. This compound is prepared by coupling two equivalents of bromoacetic acid with one equivalent of dicyclohexylcarbodiimide in an organic solvent (dioxane, CH.~CI~, CHC13, CC14). To a solution of bromoacetic acid (13.9 mg, 100 ~moles) in 0.5 ml of dry dioxane is added dicyclohexylcarbodiimide (10.5 rag, 50 ~moles). The reaction mixture is left for 1 hr at room temperature. Whenever possible, dicyclohexylurea should be removed by filtration. The bromoacetic anhydride thus formed is ready for coupling to ligands. 18To recrystallize iodoacetic acid, dissolve 5 g in 80 ml of hot cyclohexane and cool to 5° . Collect the precipitate, wash it with petroleum ether, and dry. 19D. Givol and M. Wilchek, this volume [53].