A continuous spectrophotometric direct assay for peptidyl prolyl cis-trans isomerases.
m-Nitrotyrosine incorporated into proline peptides of the general sequence -Xxx-Pro-Tyr(m-NO2)- responds to cis-trans Xxx-Pro conformational transition by changes in the pKa of its side-chain hydroxyl (Garel and Siffert, 1979). We exploited this effect to develop a continuous direct (uncoupled) assay for peptidyl prolyl cis-trans isomerases. Prior to the enzyme assay, the cis-trans equilibrium is perturbed in favor of the cis isomer by dissolving the substrate H-Ala-Ala-Pro-Tyr(m-NO2)-Ala-NH2 in a 470 mM solution of LiCl in trifluoroethanol. Upon addition of substrate to the biological buffer, the conformational equilibrium characteristic for the aqueous medium is restored, and the Ala-Pro isomerization is monitored spectrophotometrically.
García-Echeverría
C
C
School of Pharmacy, University of Wisconsin-Madison 53706.
Kofron
J L
JL
Kuzmic
P
P
Rich
D H
DH
D000591:Amino Acid Isomerases / Q000032:analysis* / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D003524:Cyclosporins / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D019696:Peptidylprolyl Isomerase; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D002352:Carrier Proteins; D003524:Cyclosporins; D009842:Oligopeptides; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase