Characterisation of Norway lobster (Nephrops norvegicus) hyaluronidase andcomparison with sheep and bovine testicular hyaluronidase

The enzyme used in this study was a partially purified sample of hyaluronid ase, extracted and purified from Nephrops norvegicus (scampi) hepatopancrea s, by acetone fractionation followed by ion-exchange chromatography on Ambe rlite(R)IRA 420 and gel filtration on Sephacryl(R)S-200-HR. The optimum pH varied according to the buffer system, with highest activity being recorded at pH 5.4 in 50 mM sodium-acetate buffer. The enzyme also required NaG at a concentration of 120 mM in the final incubation mixture for optimum activ ity. Its molecular weight, estimated by gel filtration was 320 kDa and its Km value was 0.42 mg/ml using sodium-hyaluronate from bovine trachea as sub strate. The enzyme demonstrated specificity for hyaluronic acid as substrat e and showed no activity towards closely related sulphated polysaccharides, chondroitin sulphate A, B or C. On the other hand, the sulphated polysacch arides were found to inhibit scampi hyaluronidase to varying degrees. All e nzyme activity was lost on freezing the purified extract to 60 degrees C bu t addition of dimethyl-sulfoxide (1%) or bovine serum albumin prior to free zing prevented this loss. Scampi hyaluronidase was characterised and compar ed to commercially available sheep and bovine hyaluronidase. Its specific a ctivity was nearly twice as high as that of the commercial samples. However , scampi hyaluronidase was found to be more sensitive to inhibition by a ra nge of substances. Bovine and sheep hyaluronidase were, however, inhibited to a greater extent than scampi hyaluronidase, by human serum proteins. (C) 1999 Elsevier Science Ltd. All rights reserved.