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A continuous spectrophotometric direct assay for peptidyl prolyl cis-trans isomerases
C García-Echeverría1, J L Kofron, P Kuzmic
1School of Pharmacy, University of Wisconsin-Madison 53706.
Abstract:
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.