RESUMO
Hyaluronidase from fresh human serum was purified to apparent homogeneity in a two-step procedure. Potent serum inhibitors of hyaluronidase activity were removed during the course of the purification. Isolation of the enzyme was expedited by the use of a newly devised ELISA-like assay. Enzyme activity was measured by following the rates of hydrolysis of hyaluronan (HA) adsorbed onto microtiter wells. Following enzymatic digestion, the remaining HA was measured using a cartilage-derived biotinylated HA-binding protein and an avidin-peroxidase reaction. Molecular sieve chromatography yielded a doublet of proteins with apparent molecular sizes of 42 and 50 kDa. The molecular size of the major band of protein obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was 59 kDa. Under reducing conditions, however, the size increased to 72 kDa. The pH optimum of the enzyme was 3.7. Sodium chloride concentrations greater than 100 mM were inhibitory. Activity of the serum enzyme was further characterized with a new HA-substrate gel procedure. The serum enzyme activity is different from the liver-derived activity. The tissue source of this circulating enzyme is unknown.
Assuntos
Hialuronoglucosaminidase/sangue , Cromatografia , Glicosaminoglicanos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Íons , Peso MolecularRESUMO
Hyaluronic acid plays a key role in the process of wound repair. Deposition of this glycosaminoglycan polymer is in turn controlled by levels of the enzyme hyaluronidase. Hyaluronidase activity was examined in a rat incisional skin wound model comparing laser and scalpel wounds. A polyacrylamide gel electrophoresis (PAGE) hyaluronic acid substrate assay was used to detect differences in the rates of appearance, and level, of hyaluronidase activity in wound homogenates. The hyaluronidase activity in laser wounds appeared earlier, had a bimodal distribution, and increased to a higher level than that in scalpel wounds. The origin of hyaluronidase is not clear, but control of its appearance and modulation of its activity may be a more complex process than previously assumed.
Assuntos
Procedimentos Cirúrgicos Dermatológicos , Hialuronoglucosaminidase/metabolismo , Terapia a Laser , Instrumentos Cirúrgicos , Cicatrização/fisiologia , Animais , Eletroforese em Gel de Poliacrilamida , Ácido Hialurônico/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Pele/enzimologiaRESUMO
Hyaluronic acid (HA) is a key structural element of the extracellular matrix. Turnover rates of HA are determined in part by hyaluronidases, that are themselves modulated by hyaluronidase inhibitors. A substrate polyacrylamide gel electrophoresis procedure is described here that separates enzyme from inhibitors. The HA is embedded in the gel, and following electrophoretic separation, enzymatic digestion of the HA is allowed to occur. The gel is stained with Alcian blue and can be secondarily stained with Coomassie blue. Enzymatic activities appear as cleared bands on a light blue background, while major proteins appear as dark blue bands. The procedure can be performed in the presence or absence of sodium dodecylsulfate, though levels of hyaluronidase activity decrease when the detergent is used. Hyaluronidases active in the neutral or acid pH range can be detected. This technique will facilitate characterization of hyaluronidases and inhibitors from a wide variety of sources.