Studies on the metal-mediated interactions of carboxypeptidase G2 and after proteins with triazine dyes

Hughes, Peter (1984) Studies on the metal-mediated interactions of carboxypeptidase G2 and after proteins with triazine dyes. University of Southampton, Doctoral Thesis.

Abstract

Low concentrations of divalent metal ions, particularly those of the first row transition series for example, Zn +, Cu +, Co +, Mn +, and Ni + were shown to promote the binding of a number of proteins to immobilised triazine dyes. In particular, Zn + enhanced the binding of carboxypeptidase G-2, alkaline phosphatase, yeast hexokinase, and mushroom tyrosinase to immobilised Procion red H-86N, Procion yellow H-A, Cibacron blue F3G-A and Procion blue HE-RD respectively. The binding of ovalbumin to immobilised Cibacron blue F3G-A aad Procion orange MX-G was also selectively enhanced in the presence of Al +. Metal-ion bound proteins could subsequently be eluted by the application of selected chelating agents. Using immobilised Procion red H-85N, the phenomonen was applied to the large scale purification of carboxypeptidase G-2. This resulted in the production of homogeneous enzyme as demonstrated by SDS polyacrylamide gel electrophoresis. This represents a considerable improvement over the previously employed purification protocol which produced enzyme of approximately 38% homogeneity.The nature of metal ion interactions with proteins and triazine dyes was also investigated. Difference spectroscopy and enzyme inactivation studies with Cibacron blue F3G-A and yeast hexokinase suggest the formation of a highly specific ternary complex involving enzyme, dye, and metal ion at the active site region of the enzyme. These results correlate well with the effects of metal ions in promoting the binding of hexokinase to immobilised Cibacron blue F3G-A. Inactivation studies with carboxypeptidase G-2 using a number of reactive analogues of Procion red H-SBN have suggested that binding is dependent on the high density of electrons associated with the azo bond of the dye and its neighbouring sulphonic acid residues in the formation of a protein: metal-ion: dye co-ordination complex. As Procion red MX-8B competitively inactivates and displays high affinity for carboxypeptidase G-2 in the presence of Zn +, its spectral properties were utilised as a visual affinity label for the enzyme. Proteolytic cleavage and resolution of the resulting peptides by reverse phase high pressure liquid chromatography (HPLC) have resulted in the identification of the dye binding domain of the enzyme by amino acid sequence analysis.

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