Studies on the nature of transition-metal-ion-mediated binding of triazine dyes to enzymes. The interaction of procion red MX-8B with carboxypeptidase G-2.
Several reactive azoic dichlorotriazinyl dyes specifically and irreversibly inactivate the folate-degrading enzyme carboxypeptidase G-2 at a site competitive with the enzyme substrates methotrexate (4-amino-N10-methylfolic acid) and p-aminobenzoyl-L-glutamate. Although the less reactive monochlorotriazinyl dye, Procion red H-8BN, is unable to inactivate the enzyme, it is capable of marked inhibition of inactivation by dichlorotriazinyl dyes in the presence of Zn2+. Zinc ions and, to a lesser extent other first row transition metal ions, significantly enhance the affinity of Procion red H-8BN and its analogues Procion red MX-8B and Procion red MX-2B, for carboxypeptidase G-2. It is proposed that this effect is mediated through the formation of a specific tetracoordinate Zn2+ complex between the azo linkage and adjacent sulphonate and hydroxyl functions of the dye and an appropriate ligand on the protein. Carboxypeptidase G-2 quantitatively inactivated with the dichlorotriazinyl dye, Procion red MX-8B, contains approximately 1 mol dye/mol subunit of Mr 42000. Proteolytic cleavage of the labelled enzyme and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a principal red peptide which on amino acid sequence analysis results in the identification of the dye binding domain. The affinity label, Procion red MX-8B, is believed to be attached to the hydroxylic side chain of Thr-279.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D011485:Protein Binding; D013379:Substrate Specificity; D014227:Triazines / Q000494:pharmacology; D011623:gamma-Glutamyl Hydrolase / Q000032:analysis*
D000345:Affinity Labels; D004396:Coloring Agents; D008670:Metals; D014227:Triazines; C026429:Procion Brilliant Red H8-BS; D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase