Correlation of reductimetric and turbidimetric methods for hyaluronidase assay.

When hyaluronidases act on isolated (as distinct from native) sodium hyaluronate, three changes in the properties of the substrate solution can be measured: (1) the decrease in viscosity, (2) the loss of ability to form insoluble protein salts on acidification, (3) the increase in reducing sugar content.’ Each of these changes is the basis for a quantitative method of measurement of enzyme activity, and the various methods are all currently employed by workers in the field. The changes listed are often attributed to successive phases of activity of the enzyme on the substrate (1) and the lack of concordance of results obtained with the various methods is sometimes thought to be a reflection of this heterogeneous mechanism. From a chemical standpoint, an interpretation of the mechanism of enzyme action resulting in the increase in reducing sugar is the least ambiguous: the reducing sugar is the result of the breaking of the glucosidic bonds between the alternating N-acetylhexosamine and glucuronic acid residues which constitute hyaluronic acid. There is also evidence based on the increase in the color formed with p-dimethylaminobenzaldehyde that the first bond which is broken is that which liberates the reducing group of the N-acetylhexosamine. The basis for consideration of different mechanisms has been in large measure due to the fact that the properties both of viscosity and turbidity formation with acidified protein were lost before an increase in reducing sugar could be measured. By application of the recently developed ultramicromethod for reducing sugar of Park and Johnson (2), it is possible to observe that under the conditions employed for turbidimetric assay of hyaluronidase (3), namely the action of 1 turbidimetric unit of testicular enzyme on 200 y of pure hyaluronic acid in 1.0 ml. of acetate buffer for 30 minutes at 37’ and pH 6, reducing sugar equivalent to 1.6 y of glucose is liberated. This represents 2 per cent of the theoretical