Affinity labelling 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.
Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion blue MX-R, a dichlorotriazinyl structural analogue of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37 degrees C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 microM. 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 Mr 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: (formula; see text) 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.
D000345:Affinity Labels; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004396:Coloring Agents / Q000494:pharmacology*; D006736:Horses; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification*; D014227:Triazines / Q000494:pharmacology*
D000345:Affinity Labels; D004396:Coloring Agents; D010446:Peptide Fragments; D014227:Triazines; C036598:procion blue MX-R; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase