ANALYTICAL
BIOCHEMISTRY
A Tritium
78,406-409
(1977)
Exchange Method for Characterization Modified Histidyl Residues and Its Application to Fumarase
of
GARY A. ROGERS Molecular
Biology Institute, University of California, Los Angeles. California 90024
Received July 1, 1976; accepted November 5, 1976 A technique using tritium incorporation into the C-2 position of the imidazolyl ring of histidine for the identification of alkylated histidyl residues is reported. The first-order rate constant for the incorporation of tritium from water into N-methylhistidine (pH S.O,SO”C) is 3.3 x 10~* min-’ (tt = 21 min). No other amino acid tested gave more than 0.3% as much incorporation of tritium as did N-methylhistidine. A modified amino acid residue derived from fumarase which had been inactivated by the bifunctional reagent 3,4-bis(bromomethy1) benzoate (I) was conclusively identified as a histidine-I adduct by the above characterization procedure. The technique of measuring tritium incorporation allows the qualitative identification of compounds as histidyl (or imidazolyl) or, alternatively, the quantitative determination of known histidyl compounds, both at picomole levels.
The number of enzymes which have been found to contain histidine at their active sites is indeed large. In the past, identification of this particular amino acid has rested on a variety of techniques including photoxidation (1) and alkylation by enzyme-specific (2) and nonspecific (3) reagents, followed by chromatographic analysis or product structure elucidation (3a). Alkylation of the histidyl imidazolyl nitrogen (either N-l or N-3) by common reagents such as the a-haloacetates or acetamides is easily determined by synthesis of the two possible products and chromatographic comparison with the unknown. It is also sometimes possible to detect the absence of one histidyl residue per mole of protein and, hence, infer that a modified histidine has been generated. If an enzyme catalyzes the alkylation of a histidyl residue by an otherwise unreactive reagent, or if the reagent possesses multiple, nonequivalent sites of reactivity, then, only product structure elucidation or a difference chromatogram for modified vs unmodified protein is possible or practical for determining the identity of the modified amino acid. In the present study involving the alkylation of several amino acids at or near the active site of fumarase by the reagent 3,4-bis (bromomethyl) benzoate (I), none of the techniques just mentioned could be applied to 406 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OW3-2697
HISTIDYL
IDENTIFICATION
WITH
TRITIUM
407
determine the identity of an alkylated amino acid suspected to be histidine (4). There are four possible 1: 1 covalent adducts (as well as eight possible 1:2 and 2: 1 adducts) ofl-histidine and compoundz. Attempts to prepare all or any of these compounds in both aqueous and organic solvents failed because of the inherent low reactivity of Z with nitrogen nucleophiles (5). Furthermore, because fumarase contains 54 histidyl residues per molecule, loss of only 2 or 3 from the chromatogram due to modification would be near the limits of detectability by present analytical techniques. A simple procedure which obviates these difficulties and is suitable for the detection and quantitation of picomole amounts of histidine or its derivatives has been developed and is reported herein. Inactivation of fumarase with 3H-labeled Z followed by acid hydrolysis and column chromatography on anion-exchange resin (AGl-X8) with elution by pH gradients gave four radioactive fractions. Elution of one fraction (about 25% of total radioactivity recovered) at pH 7 suggested that it might contain modified histidine (because of additional carboxylate introduced by Z). However, the Pauli test would fail to identify the fraction as containing histidine because diazo coupling does not occur with N-substituted imidazoles (6). A procedure involving the facile exchange (Ref. (7), Eq. [I]) of the C-2 proton of imidazolyl groups with solvent protons was selected as a possible means of confirming the identity of the modified amino acid as histidine. First, the technique was tested on a group of alkylated amino acids and a tripeptide in order to determine if other protons would exchange with rates comparable to the C-2 proton of histidine.
3-Methylhistidine was used as a model for an alkylated histidyl residue which might be derived from protein modification studies. 3-Methylhistidine (0.894 pmol) was dissolved in 1.1 ml of Tris buffer (60 mM, pH 8.0) containing 3H,0 (27.8 mCi/ml). Multiple samples were placed in a heating block at 80 + 1°C and were quenched at various reaction times by immersion into an ice bath followed by addition of 50 ~1 of concentrated HCl. Each sample was chromatographed separately on 0.7-ml Dowex AG 50-X4, and a radioactive peak eluted coincidently with 3-methylhistidine. The rate of incorporation of tritium was first-order with kobs = 3.3 x 1OV min-l ( f1 = 21 min). Table 1 shows results for a number of amino acids and
408
GARY A. ROGERS TABLE TRITIUM
INCORPORATION
Compound N-Methylhistidine S-Ethylcysteine O-Methyltyrosine O-Methylserine Tyrosine Tryptophan GSH
1
INTOALKYLATEDAMINOACIDSAND
3H incorporation (cpminmol) 353 so. I -0.82 1.08 co.4 Cl -0.85
GSH” Relative incorporation I
BIOCHEMISTRY
A Tritium
78,406-409
(1977)
Exchange Method for Characterization Modified Histidyl Residues and Its Application to Fumarase
of
GARY A. ROGERS Molecular
Biology Institute, University of California, Los Angeles. California 90024
Received July 1, 1976; accepted November 5, 1976 A technique using tritium incorporation into the C-2 position of the imidazolyl ring of histidine for the identification of alkylated histidyl residues is reported. The first-order rate constant for the incorporation of tritium from water into N-methylhistidine (pH S.O,SO”C) is 3.3 x 10~* min-’ (tt = 21 min). No other amino acid tested gave more than 0.3% as much incorporation of tritium as did N-methylhistidine. A modified amino acid residue derived from fumarase which had been inactivated by the bifunctional reagent 3,4-bis(bromomethy1) benzoate (I) was conclusively identified as a histidine-I adduct by the above characterization procedure. The technique of measuring tritium incorporation allows the qualitative identification of compounds as histidyl (or imidazolyl) or, alternatively, the quantitative determination of known histidyl compounds, both at picomole levels.
The number of enzymes which have been found to contain histidine at their active sites is indeed large. In the past, identification of this particular amino acid has rested on a variety of techniques including photoxidation (1) and alkylation by enzyme-specific (2) and nonspecific (3) reagents, followed by chromatographic analysis or product structure elucidation (3a). Alkylation of the histidyl imidazolyl nitrogen (either N-l or N-3) by common reagents such as the a-haloacetates or acetamides is easily determined by synthesis of the two possible products and chromatographic comparison with the unknown. It is also sometimes possible to detect the absence of one histidyl residue per mole of protein and, hence, infer that a modified histidine has been generated. If an enzyme catalyzes the alkylation of a histidyl residue by an otherwise unreactive reagent, or if the reagent possesses multiple, nonequivalent sites of reactivity, then, only product structure elucidation or a difference chromatogram for modified vs unmodified protein is possible or practical for determining the identity of the modified amino acid. In the present study involving the alkylation of several amino acids at or near the active site of fumarase by the reagent 3,4-bis (bromomethyl) benzoate (I), none of the techniques just mentioned could be applied to 406 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN OW3-2697
HISTIDYL
IDENTIFICATION
WITH
TRITIUM
407
determine the identity of an alkylated amino acid suspected to be histidine (4). There are four possible 1: 1 covalent adducts (as well as eight possible 1:2 and 2: 1 adducts) ofl-histidine and compoundz. Attempts to prepare all or any of these compounds in both aqueous and organic solvents failed because of the inherent low reactivity of Z with nitrogen nucleophiles (5). Furthermore, because fumarase contains 54 histidyl residues per molecule, loss of only 2 or 3 from the chromatogram due to modification would be near the limits of detectability by present analytical techniques. A simple procedure which obviates these difficulties and is suitable for the detection and quantitation of picomole amounts of histidine or its derivatives has been developed and is reported herein. Inactivation of fumarase with 3H-labeled Z followed by acid hydrolysis and column chromatography on anion-exchange resin (AGl-X8) with elution by pH gradients gave four radioactive fractions. Elution of one fraction (about 25% of total radioactivity recovered) at pH 7 suggested that it might contain modified histidine (because of additional carboxylate introduced by Z). However, the Pauli test would fail to identify the fraction as containing histidine because diazo coupling does not occur with N-substituted imidazoles (6). A procedure involving the facile exchange (Ref. (7), Eq. [I]) of the C-2 proton of imidazolyl groups with solvent protons was selected as a possible means of confirming the identity of the modified amino acid as histidine. First, the technique was tested on a group of alkylated amino acids and a tripeptide in order to determine if other protons would exchange with rates comparable to the C-2 proton of histidine.
3-Methylhistidine was used as a model for an alkylated histidyl residue which might be derived from protein modification studies. 3-Methylhistidine (0.894 pmol) was dissolved in 1.1 ml of Tris buffer (60 mM, pH 8.0) containing 3H,0 (27.8 mCi/ml). Multiple samples were placed in a heating block at 80 + 1°C and were quenched at various reaction times by immersion into an ice bath followed by addition of 50 ~1 of concentrated HCl. Each sample was chromatographed separately on 0.7-ml Dowex AG 50-X4, and a radioactive peak eluted coincidently with 3-methylhistidine. The rate of incorporation of tritium was first-order with kobs = 3.3 x 1OV min-l ( f1 = 21 min). Table 1 shows results for a number of amino acids and
408
GARY A. ROGERS TABLE TRITIUM
INCORPORATION
Compound N-Methylhistidine S-Ethylcysteine O-Methyltyrosine O-Methylserine Tyrosine Tryptophan GSH
1
INTOALKYLATEDAMINOACIDSAND
3H incorporation (cpminmol) 353 so. I -0.82 1.08 co.4 Cl -0.85
GSH” Relative incorporation I
