Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.
We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.
Toida
T
T
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Chiba, Inage, 2638522, Japan. toida@p.chiba-u.ac.jp
Ogita
Y
Y
Suzuki
A
A
Toyoda
H
H
Imanari
T
T
D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005069:Evaluation Studies as Topic; D017022:Flow Injection Analysis / Q000379:methods*; D005456:Fluorescent Dyes; D006025:Glycosaminoglycans / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000652:urine; D009682:Magnetic Resonance Spectroscopy; D013451:Sulfonic Acids / Q000737:chemistry
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; D013451:Sulfonic Acids; D006821:Hyaluronoglucosaminidase