Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates.
Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).
Szabelski
M
M
Faculty of Chemistry, University of Gdańsk, Poland.
Stachowiak
K
K
Wiczk
W
W
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D016166:Free Radical Scavengers; D010455:Peptides; D010206:Papain; D004121:Dimethyl Sulfoxide