An improved assay for the N-acetyl-D-glucosamine reducing ends of polysaccharides in the presence of proteins.

Hyaluronan concentration and hyaluronidase activity can be assayed by using different techniques including turbidimetry, viscosimetry, ELISA, chromatography, and colorimetry. The most popular colorimetric method is that of J. Reissig et al. (1955, J. Biol. Chem. 217, 959-966), in which the color results from a reaction between the Ehrlich's reagent (DMAB) and the N-acetyl-d-glucosamine reducing end of each hyaluronan chain. Nevertheless, there are problems with this method when proteins are present in the medium. Here we propose a new interpretation of the Reissig signal for estimating such reducing ends in media containing enzymes or other proteins. This interpretation is based on the fact that the absorbance obtained by using the Reissig method results from two factors: a turbidity due to the formation of polysaccharide-protein complexes and a color resulting from the action of DMAB on the reducing end of the polysaccharide chains. The turbidity at 585 nm, the wavelength at which the color intensity is maximal, may be estimated by curve fitting the spectrum between 450 and 650 nm. Subtracting the turbidity from the absorbance gives the colorimetric intensity which represents the concentration of polysaccharide chains. Moreover, the turbidity may give additional information about the existence of polysaccharide-protein complexes and their nature.

Kinetic study of the alpha-chymotrypsin-catalyzed hydrolysis and synthesis of a peptide bond in a monophasic aqueous/organic reaction medium.

We have studied the hydrolysis and synthesis reactions of the peptide bond involved in N-Cbz-L-tryptophanyl-glycineamide as catalyzed by alpha-chymotrypsin in various mixtures of water and 1,4-butanediol. Using a constant nonsaturating concentration of substrate, the initial reaction rates decreased exponentially with decreasing water content in the solvent mixture. When the water content was decreased from 100 to 20% (v/v), the maximum rate of reaction did not vary by more than a factor of 2 to 4, whereas the Michaelis constant increased exponentially. This exponential variation of the Michaelis constant was due to changes in the partitioning of the substrate between the active site of the enzyme and the solvent. In these processes, the actual rate constants did not vary when the relative contents of water and 1,4-butanediol were varied.

Gram-scale enzymatic synthesis of a peptide bond.

The synthesis reaction of the peptide, N-Cbz-L-tryptophanyl-glycineamide, catalyzed by alpha-chymotrypsin was performed in a 20% water/80%, 1,4-butanediol mixture. The synthesis yield reached 90.9% at the end of the reaction and 72.3% after purification. The effects on the yield of both pH and the ratio between total initial concentrations of glycineamide and N-Cbz-L-tryptophan are examined. The high yield, specificity, simplicity and reproducibility of this method make it complementary of the chemical methods.