pH controlled diazo coupling of aldolase. Selective formation of diazothioether chromophores and retention of enzyme activity.

pH Conditions have been found which achieve selective reaction of diazotized p-amino benzoate with cysteine residues of rabbit muscle aldolase. The difference in reactivity of the two sulphydryl groups involved, (Cys--237 and Cys--287) permits one to form either four or eight diazothioethers on the tetrameric enzyme and obtain a homogeneous protein. In both cases the enzyme became slightly more active in the fructose-1, 6-bisphosphate cleavage, the KM value being retained. The results have been discussed with regard to chemically modifying an enzyme to change its physical, chemical and immunological properties, whilst leaving the catalytical activity unmodified.

Catalytically active azoaldolase. Preparation on solid support.

A procedure for the coupling at pH 7.2 of p-carboxy benzene diazonium chloride with rabbit muscle aldolase supported on phosphocellulose is described and some of the spectroscopic, structural and catalytic features of the material obtained are reported. The tetrameric azoenzyme is homogeneous in disc gel electrophoresis even in the presence of 8 M urea. Twelve molecules of the reactant are bound to the protein. Eight azocysteins are identified by both spectroscopic studies and amino acid analysis. The presence of one azohistidine is suggested by the spectroscopic data along with the presence of other, as yet unknown, chromophores. The azoaldolase shows unchanged catalytic properties using both D-fructose 1,6-bisphosphate and D-fructose 1-phosphate as substrates, as compared with the native enzyme. The pH profile of the enzyme activity is broadened towards the alkaline region but no changes occur in the physiological range of pH.