Colorimetric determination of hyaluronidase activity.

Turbidimetric methods for assay of hyaluronidase activity are based upon the measurement of the optical density of suspensions of substrateprotein combination in an acid environment (1). The reliability of this measurement depends upon careful control of the conditions under which turbidity develops (2), and even then the relation between substrate concentration and optical density is linear over only a small range. The introduction of disodium phenoltetrabromophthalein (bromosulfalein, Hynson, Westcott and Dunning, Inc.) for the determination of the amount of protein combined with substrate (3) permits measurement of a relatively stable color instead of a changing turbidity, and widens the range of substrate concentrations that can be tested. Materials and ReagentsBu$ers-pH 2.5 citrate-phosphate according to McIlvaine (4), to which 5.7 moles per liter of urea are added. Acetate (pH 6.0) 0.1 M containing 0.15 M NaCl. Plasma #o&ion-Human blood specimens collected in acid-citrate-dextrose solution and discarded from a blood bank proved satisfactory as a plasma source. The plasma is diluted 1: 10 with the buffer of pH 2.5. Cuvettes-Test-tubes without lips 15 X 100 mm. (Arthur H. Thomas, No. 9446) are tested for uniformity in a spectrophotometer with CuS04.5Hz0, 15 gm. per 100 ml. of HzO, and a wave-length of 560 rnp. Tubes which read within an optical density variation of *0.002 are marked to contain 10 ml. Xpectrophotometer-Coleman junior, model 6A. Centrifuge-International centrifuge size 1, type SB, with angle head No. 811. Xyringes-1 ml. and 0.25 ml. tuberculin type, and 3 ml. standard glass type. Water Bath-Regulated to maintain temperature at 37.5 f 0.1”. Bromosulfalein Xolution-The contents of a 3 ml. ampul of 5 per cent aqueous solution are added to 197 ml. of the above buffer, pH 2.5. Substrate-Potassium hyaluronate prepared from human umbilical cords by the method of Byers et al. (5) (found on analysis: 2.4 per cent nitrogen,