AFFINITY LABELING OF ENZYMES WITH TRIAZINE DYES - ISOLATION OF A PEPTIDE IN THE CATALYTIC DOMAIN OF HORSE-LIVER ALCOHOL-DEHYDROGENASE USING PROCION BLUE MX-R AS A STRUCTURAL PROBE

  • 1 January 1982
    • journal article
    • research article
    • Vol. 128  (1) , 119-123
Abstract
Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion Blue MX-R, a dichlorotriazinyl structural analog of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37.degree. C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 .mu.M. Methoxylated Procion blue MX-R does not inactivate the enzyme. The inactivation of horse liver alcohol dehydrogenase by Procion blue MX-R is competitively inhibited by the pyridine nucleotides NAD+ and NADH. Quantitatively inhibited horse liver alcohol dehydrogenase contains 1 mol dye/mol subunit of MW 40,000. Chymotryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a single blue peptide which on sequencing and analysis yields an amino acid sequence of Ile172-Gly173-Cys174-Gly175-Phe176 with the affinity label, Procion blue MX-R, unambiguously identified as being attached to the thiol side chain of Cys-174 in the catalytic domain of the enzyme. The specific active site-directed reaction of Procion blue MX-R with horse liver alcohol dehydrogenase is interpreted in terms of the known crystallographic structure of the enzyme.