A turbidimetric method for the assay of hyaluronidase.

A variety of methods have been devised for the assay of the enzyme hyaluronidase. These may be divided into four types: (a) measurement of the “spreading effect” in the skin of rabbits and guinea pigs (1, 2), (b) reduction of viscosity (3,4), (c) prevention of mucin clot formation (5), and (d) chemical measurement of total reducing substances, N-acetylglucosamine, or glucuronic acid liberated on the complete hydrolysis of hyaluronic acid (6, 7). The first group of methods (“spreading activity”) is most sensitive, but is time-consuming, requires large numbers of animals, and lacks specificity in that substances other than hyaluronidase have been shown to possess spreading activity. They are of comparatively little value for the routine assay of large numbers of preparations. The viscosity method has been used widely with considerable success. It is, however, tedious and timeconsuming and requires large amounts of hyaluronic acid. The original mucin clot prevention method is based,on the fact that hyaluronic acid reacts with acidified albumin to form a clot (5). If hyaluronic acid is first acted on by hyaluronidase, such a clot does not form. This then affords a basis for the estimation of hyaluronidase. It has been noted that when the smallest amount of enzyme necessary to prevent clot formation is used there remains a uniform turbidity. If larger amounts of enzyme are used, this turbidity does not develop. The use of the various chemical met,hods is open to serious question in that Hahn (8) has recently shown that highly purified hyaluronidase does not bring about the complete hydrolysis of hyaluronic acid. The release of glucuronic acid and N-acetylglucosamine apparently depends upon the presence of contaminating enzymes in the crude preparations. A method based on turbidity development seemed most likely to be rapid, simple, and accurate. Seastone (9) showed that under certain conditions turbidity is directly proportional to hyaluronic aeid concentration. Kass and Seastone (10) used this fact in developing a quantitative turbidimetric method for the assay of hyaluronidase. They utilized acidified serum for turbidity development. Leonard, Perlman, and Kurzrok (11) have developed a similar method for the determination of hyaluronidase in semen by use of acidified serum.