Abstract
Human carbonic anhydrase C and bovine carbonic anhydrase B were modified with the affinity label, bromoacetazolamide, and human carbonic anhydrase B with N-bromoacetylacetazolamide. Tryptic peptides from the modified enzymes, containing alkylated histidines, have been isolated by ion exchange and two-dimensional high voltage electrophoresis-paper chromatography. The partial amino acid sequence of the peptide from alkylated bovine enzyme B has been found to be Met-Val-Asn-Asn-Gly-His-Ser-Phe-Asn-Val-Glu-Tyr-Asx-Asx(Glx,Asx,Ser)Lys-. In all probability the histidine in this sequence that reacted is His-64. Comparison of the amino acid composition of the tryptic peptides from the two human isoenzymes with the proposed sequences of these enzymes has made it possible to identify histidine-64 in human enzyme C as that reacting with bromoacetazolamide and histidine-67 in human enzyme B as that reacting with N-bromoacetylacetazolamide.
Highlights
Comparison of the amino acid composition of the tryptic peptides from the two human isoenzymes with the proposed sequences of these enzymes has made it possible to identify histidine-64 in human enzyme C as that reacting with bromoacetazolamide and histidine-67 in human enzyme B as that reacting with with histidine-2001 in human carbonic anhydrase B (9) ; the sequentially homologous amino acid in human enzyme C is leucine (10)
In this communication we report the isolation of tryptic peptides containing an alkylated histidine from human carbonic anhydrase C and bovine carbonic anhydrase B alkylated with bromoacetazolamide, as well as the isolaticn of a tryptic peptide from human carbonic anhydrase B alkylated with N-bromoacetylacetaaolamide
Alkylated bovine enzyme B was separated from unreacted enzyme by t,he use of DEAE-cellulose chromatography as reported earlier (12) (Fig. 1)
Summary
Materials and Reagents-Human and bovine carbonic anhydrases were isolated as reported earlier (12). Bromo[14C]acetazolamide, with a specific activity of 0.35 &i per pmole, and N-. Bromo[14C]acetylacetazolamide, with a specific activity of 0.16. PC1 per pmole, were prepared in a manner similar to that already described (3, 12). Trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone, three times recrystallized chymotrypsin, and carboxypeptidase A, both treated with diisopropylphosphoro-fluoridate, were obtained from Worthington. Dowex 50 resin (AG 5OWX2, 200 to 400 mesh), Dowex 1 resin (AG l-X2, 200 to 400 mesh) and DEAE-cellulose (Cellex D, high capacity) were obtained from Bio-Rad, California. L’henyl isothiocyanate, trifluoroacetic acid, and pyridine for sequential degradation (sequential grades) were purchased from Pierce. All other solvents were reagent grade and were redistilled before use
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