Purification and Properties of Ovhe Testicular Hyaluronidase. D. B. MORTON. Tissue Physiology Department, Strangeways Research Laboratory, Cambridge.
534th MEETING, NOTTINGHAM
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Purification and Properties of Ovhe Testicular Hyaluronidase D. B. MORTON Tissue Physiology Department, Strangeways Research Laboratory, Cambridge CB1 4RN, U.K. Hyaluronidase (EC 3.2.1.35) was purified from commercial ovine testicular hyaluronidase (type 11;Sigma Chemical Co., St. Louis, Mo., U.S.A.) as part of an immunoenzymic study on enzymes in the acrosomal region of spermatozoa. The enzyme was found to exist as two isoenzymes with isoelectric points of about 6.4 and 9.1, as judged by prepafative isoelectric-focusing columns. The isoenzyme with the higher PI (which was responsible for 65 % of activity in the crude material) was purified some 120-fold and has a final specific activity of 7.47 units/mg of protein, 1 unit being defined as 1pmol of N-acetylglucosamine released/min under the assay conditions used. The yield of activity was 25 %. Purification was carried out on DEAE-cellulose, CM-cellulose (Whatman), hydroxyapatite and Sephadex G-150. Final preparations were not homogeneous and consisted of at least two proteins, as judged by polyacrylamide isoelectric focusing and Ouchterlony immunodiffusion plates. The molecular weight of the purified enzyme was found to be about 70000. Monospecific antisera to hyaluronidase have been raised in three rabbits by injecting antibody-antigen precipitin lines from immunodiffusion plates. The antisera completely inhibited the hyaluronidase activity due to both isoenzymes, and were also found to cross-react with bovine hyaluronidase. I thank the Wellcome Trust and the Agricultural Research Council for their support.
Photoconductionin Dry Cytochrome c Oxidase Preparations D. D. ELEY and E. METCALFE Department of Chemistry, University of Nottingham, Nottingham NG7 2RD, U.K. and R. J. MAYER Department of Biochemistry, University of Nottingham Medical School, Nottingham NG7 2RD, U.K. The study of photoconduction in cytochrome c oxidase, where electrons are injected by photons, may throw light on the enzyme mechanism in the respiratory chain, where electrons are injected by substrate. A practical problem is to obtain suitably pure enzyme preparations. Three samples have been prepared by the method of Yonetani (1967): for sample I, enzyme solution (12mg/ml) in 0.1 M-potassium phosphate buffer, pH 7.4, containing 2% (w/v) of potassium cholate was salted out at 35% saturation with (NH4)2S04and the precipitate was freeze-dried to give a green powder; for sample 11, enzyme solution (6mg/ml) in 0.1 M-potassium phosphate buffer, pH 7.4, containing 1% (w/v) of Tween 80 (Sigma Chemical Co., St. Louis, Mo., U.S.A.) was freezedried to give a slightly oily specimen; for sample 111, enzyme solution (10mg/ml) in 0.1 M-potassium phosphate buffer, pH7.4, containing 1 % (w/v) of Tween 80 was dialysed against 0.25~-sucroseadjusted to pH7.4 with NaHC03; the enzyme in a relatively pure highly aggregated form was thereafter precipitated by centrifugation at 10000Og,,. for 2h. Direct-current electrical conductivities were measured on pressed discs, thickness 0.345mm (sample I), 0.08mm (sample 11) and 0.12mm (sample 111), sandwiched between transparent conducting quartz electrodes and dried in a vacuum of 1mPa. Results for dark conduction and photoconduction in these preparations, together with data on the main impurities, (NH4)2S04and Tween 80 (cholate and sucrose are omitted as they are relatively good insulators) are shown in Table 1. After due regard to time-constants (resistance x capacity) and electron-trapping (Eley & Pacini, 1968), we have classified the charge carriers as electronic if equilibrium currents are set up VOl. 1
Table 1 . Electrical data for cytochrome oxidase and its major impurities Property Probable carriers ... Approx. conductivity (u) at 300°K
Sample 1 Ions (
