A quenched fluorescent dipeptide for assaying dispase- and thermolysin-like proteases.
Metalloproteases such as dispase and thermolysin play a crucial role in the life cycle of bacteria. Commonly, they prefer hydrophobic amino acids at P1' of substrate proteins, thereby cleaving the peptide bond at the alpha amino group. Activity of such proteases has been measured by the use of tailor-made oligopeptides provided with fluorescence resonance energy transfer dyes. We can now show that the short dipeptide Dabcyl-Ser-Phe-EDANS is an appropriate substrate of dispase and thermolysin. It was cleaved by both enzymes at the single peptide bond accompanied by a steep increase in fluorescence. Substantial quenching effects of the formed products were observed only when more than 80microM substrate was hydrolyzed. High affinity of the proteases for the dipeptide resulted in low K(m) values of 91+/-9 and 104+/-18microM, which are comparable to those measured for longer peptides. Dabcyl-Ser-Phe-EDANS was also used to determine the pH and optimal temperature of dispase, which were found at pH 7.0 and 50 degrees C. Buffer substances such as acetate, citrate, and tris(hydroxymethyl)aminomethane had no significant effect on enzyme activity. Measurements up to 100 degrees C revealed that hydrolysis of the quenched fluorescent dipeptide took place only in the presence of active dispase.
Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, D-64287 Darmstadt, Germany.
Stefanie
S
Weimer
Kai
K
Oertel
Hans-Lothar
HL
Fuchsbauer
D001665:Binding Sites; D004151:Dipeptides / Q000138:chemical synthesis / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K / Q000378:metabolism / Q000494:pharmacology; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
C512304:Dabcyl-Ser-Phe-EDANS; D004151:Dipeptides; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010450:Endopeptidases; D019286:Endopeptidase K; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; C023139:dispase