David G. Herries

The structure of porphyrins and haems in aqueous solution

Abstract Dimerization occurs extensively in aqueous solutions of protoferrihaem and related ferrihaems with substitutions at the 2, 4 positions. The dimerization constants for protoferrihaem (K = 4.5), deuteroferrihaem (K = 0.034), mesoferrihaem (K = 0.0692), haematoferrihaem (K = 0.010) and coproferrihaem (K = 0.0021) varied with the hydrophobicity of the 2, 4 substituents except for protoferrihaem, where additional π-Π bonding, due to the vinyl groups, was indicated. Dimerization may have important implications for the formation of ferrihaems in vivo and for studies of ferrihaems in vitro.

The multiple forms and kinetic differences of rat colonic β-N-acetylhexosaminidases

Rat colonic beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been separated into three forms by DEAE-cellulose chromatography with an increasing salt gradient. It was not possible to separate the glucosaminidase activity from the galactosaminidase activity by a variety of chromatographic procedues, but the ratio of the two specific activities varied during purification. The pH optima were however identical, for both activities and all three forms. Kinetic measurements including inhibition by substrate analogues showed differences between the two activities as well as among the three forms. A common active site model was inconsistent with the results. Data from mixed substrate experiments were consistent with a model wherein the two activities reside in seperate active sites, each able to be inhibited by the substrate for the other site. The effect of acetate and SH reagents confirmed the two-site model. Treatment with neuraminidase, thimerosal, p-hydroxymercuribenzoate, HgCl2 and AgNO3 or heating at 50 degrees C did not produce any effect on the A form that could be identified as a conversion to the B form. Measurement of the effects on both activities supported the two-site model. It is concluded that the relationship between the A and B forms in the rat colonic mucosa hexosaminidases must be different from that reported for such enzymes from other sources.

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.

Kinetic evidence for two types of active site in the N-acetyl-β-D-hexosaminidase of bovine kidney

N-Acetylhexosaminidases have been reported to hydrolyse the terminal N-acetylglucosamine and N-acetylgalactosamine from fl-glycosidic linkages at comparable rates. In general, it has proved impossible to separate the N-acetyl-/3-D-glucosaminidase activity (2-acetamido-2-deoxy&D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) from the N-acetyl-D-Dgalactosaminidase activity (2-acetamido-2-deoxy-flD-galactoside acetamidodeoxygalactohydrolase, EC 3.2.1 S3) by conventional protein fractionation procedures, and this fact, together with the close similarity between their pH and thermal inactivation profiles, has established the view that the two activities are carried by one enzyme that has been designated N-acetylhexosaminidase . Previous kinetic investigations, some rather limited, have concluded that the same active sites are responsible for the two activities [l-7]. However, a detailed kinetic study from this laboratory on the enzymes of rat colonic tissues strongly suggested the presence of two separate active sites, each able to be inhibited by the substrate for the other site [8]. In view of this apparent difference between the rat colonic enzymes and those reported from other sources, it seemed to us desirable to carry out a similar detailed kinetic investigation on a readily available preparation of N-acetylhexosaminidase.