Open AccessAffinity Labelling of Enzymes with Triazine Dyes
Author(s) -
SMALL David A. P.,
LOWE Christopher R.,
ATKINSON Tony,
BRUTON Chris J.
Publication year - 1982
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1982.tb06941.x
Subject(s) - alcohol dehydrogenase , chemistry , enzyme , nad+ kinase , affinity label , dehydrogenase , peptide , biochemistry , lactate dehydrogenase , enzyme assay , chromatography , stereochemistry
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°C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 μM. 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 M r 40000. 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: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.