A fluorimetric Morgan-Elson assay method for hyaluronidase activity.
Despite their physiological importance, hyaluronidases (HAases) have long been "neglected enzymes," due, presumably, in part to the lack of rapid, sensitive assays. Currently, the colorimetric Morgan-Elson assay method, which is based upon the generation of a new reducing N-acetyl-D-glucosamine terminus with each cleavage reaction, is most widely employed but is yet insensitive. We, therefore, reinvestigated the colorimetric method and established the fluorimetric Morgan-Elson assay for HAase activity, with the optimized tetraborate reagent. The fluorimetric assay, requiring neither specialized reagents nor a long time to perform, provided high sensitivity, nearly comparable to that of enzyme-linked immunosorbent assay (ELISA)-like assays, with a detection limit of 5 x 10(-3)NFU/ml of bovine testicular HAase after 1-h incubation. The increased sensitivity permitted rapid measurement of low HAase activity in biological samples such as human and rabbit serum HAases, the latter of which has not been detected either by an ELISA-like assay or by zymography. Human serum HAase was easily characterized it along with its optimum pH and kinetic parameters.
Takahashi
Tomoko
T
Institute of Medicinal Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawaku, Tokyo 142-8501, Japan. t-tomoko@hoshi.ac.jp
Ikegami-Kawai
Mayumi
M
Okuda
Ryozou
R
Suzuki
Kaori
K
D000117:Acetylglucosamine / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002417:Cattle; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D011133:Polysaccharide-Lyases / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D012680:Sensitivity and Specificity; D013737:Testis / Q000201:enzymology
D006821:Hyaluronoglucosaminidase; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase; D000117:Acetylglucosamine