A chromogenic oxidative coupling reaction of laccase: applications for laccase and angiotensin I converting enzyme assay.
A sensitive chromogenic assay for p-hydroxybenzoic acid, laccase activity, and angiotensin I converting enzyme activity is described. The method relies on the oxidative coupling of 2,2'-azino-di(3-ethylbenzothiazoline-6-sulfonic acid) and p-hydroxybenzoic acid. Lacase catalyzes the formation of a deep-purple compound, which shows a broad absorption between 530 and 630 nm with a maximum at 593 nm (the molar absorption coefficient was calculated to be 26,900). By means of this chromogenic coupling reaction, a spectrophotometric method for the assay of laccase activity and estimation of the amount of p-hydroxybenzoic acid was developed; laccase activity in the range 1-10 pmol protein could be estimated with a 10-min incubation time. Angiotensin I converting enzyme was also assayed by the laccase-catalyzed indicator reaction, using p-hydroxybenzoyl-glycyl-histidyl-leucine as the substrate, and N alpha-carbobenzoxy amino acid urethane hydrolase as the coupling enzyme.
Shin
T
T
Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
Murao
S
S
Matsumura
E
E
D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D042845:Laccase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis*; D007703:Peptidyl-Dipeptidase A / Q000032:analysis*
D010088:Oxidoreductases; D042845:Laccase; D007703:Peptidyl-Dipeptidase A