The application of triazine dyes in enzyme purification and structural studies

A number of triazine dyes, including Cibacron blue F3G-A and Procion blue MX-R, have been covalently attached to microparticulate silica and used for the resolution of proteins by high performance liquid affinity chromatography (HPLAC). The versatility of HPLAC, a technique which combines the inherent speed and resolving power of high performance liquid chromatography (HPLC) with the biological specificity of affinity chromatography, was applied to the resolution of protein mixtures containing enzymes such as lactate dehydrogenase, hexokinase, alkaline phosphatase, carboxypeptidase G and L tryptophanyl-tRNA synthetase. In addition, the effect of divalent metal ions such as Mg + and Zn + on promoting the adsorption of enzymes to silica immobilised triazine dye adsorbents has been investigated. Dye-metal ionenzyme intractions are interpreted in terms of a structure-function relationship.The preparative scale purification of rabbit muscle lactate dehydrogenase from a crude extract was achieved on a HPLAC adsorbent comprising silica immobilised Procion blue MX-R. Bound enzyme was biospecifically eluted with NADH, to yield a homogeneous protein as judged by SDS polyacrylamide gel electrophoresis. Two further HPLAC adsorbents have been developed, silica immobilised acriflavin which functions as a charge transfer matrix and silica immobilised iminodiacetic acid which operates as a metal chelate affinity chromaiography adsorbent. The acriflavin-silica adsorbent is able to resolve mixtures of purines and pyrimidines, nucleosides and nucleotides, pyridine nucleotide coenzymes, flavins and aromatic amino acids, achieving separation in a fraction of the time required for similar separations by low-pressure liquid chromatography. The metal chelate HPLAC adsorbent was applied specifically to the purification -1f the metalloenzyme, carboxypeptidase G from a crude extract (10.8 u.mg ). Chromatography yielded a substantially purified enzyme (approx. 90%) as judged by-1SDS polyacrylamide gel electrophoresis with a specific activity of 248 u.mg. The ability of the dichlorotriazinyl dye, Procion blue MX-R, to mimic coenzyme binding was exploited as an affinity label for horse liver alcohol dehydrogenase. Chymotryptic digestion and resolution of peptides yielded a single dye modified peptide which was sequenced, and the site of labelling on the enzyme identified. 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.