Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa.
A series of Glu(pNA)-containing peptides was designed to determine the activity of the transglutaminase factor XIIIa at 405 nm due to p-nitroaniline release. The most suitable substrate properties were found for peptides containing the Glu(pNA) residue in the second position from the N terminus. For the best substrate 12 (H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH(2)), a k(cat)/K(m) value of 3531 s(-1)M(-1) was found. Although the k(cat)/K(m) values of the Glu(pNA) peptides are more than 100-fold reduced compared with the previously reported cleavage of natural glutamine-containing substrates such as α(2)-antiplasmin and β-casein, these chromogenic substrates can be useful tools for convenient determination of FXIII-A(2)* activity e.g., for in vitro inhibitor screening. As an example, peptide 12 was used to characterize the inhibition of FXIII-A(2)* by the well-known irreversible inhibitor iodoacetic acid.
Institute of Pharmaceutical Chemistry, Philipps University Marburg, D-35032 Marburg, Germany.
Kornelia
K
Hardes
Gero Lutz
GL
Becker
M Zouhir
MZ
Hammamy
Torsten
T
Steinmetzer
D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D001671:Biochemistry / Q000379:methods*; D001681:Biological Assay; D001777:Blood Coagulation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D026122:Factor XIIIa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D019807:Iodoacetic Acid / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects
D002863:Chromogenic Compounds; D010455:Peptides; D026122:Factor XIIIa; D019807:Iodoacetic Acid