cyclophilin and the FK506 binding protein (FKBP) fam-Peptidyl prolyl cis/trans isomerases (PPIases) are ily are the cellular receptors of the immunosuppressive ubiquitous and abundant enzymes catalyzing peptide drugs cyclosporin A (CsA) and FK506, respectively, actbond cis/trans isomerization adjacent to proline in ing in a gain of function model in T cells (1). The enzypeptides and proteins. An uncoupled protease-free matic activity of PPIases is generally assessed by an assay of PPIase activity has been developed using the assay that is based on isomer-specific proteolysis using standard tetrapeptide substrates of the proteolytically tetrapeptide derivatives (Suc-Ala-Xaa-Pro-Yaa-(4-) nicoupled test system. Differences in the UV/vis absorptroanilides with Xaa, Yaa for any natural amino acid) tion spectra of cis and trans conformations of Suc-Alaas standard substrates (2, 3). The major part of the Xaa-Pro-Phe-(Y-) anilide (Xaa Å Ala, Leu, Phe; Y Å 4reported kinetic constants of PPIase catalysis has been nitro, 2,4-difluoro) were exploited to monitor the time evaluated with this type of proline peptides. Due to the course of the cis/trans isomerization subsequent to a quasi-irreversibility of the prolyl isomerization under solvent jump from 0.47 M LiCl/trifluoroethanol into these conditions, the proteolytically coupled test peraqueous solution. The utility of the assay has been mits the calculation of data for cis prolyl bonds 2 only. demonstrated by the determination of the Michaelis-Another assay used routinely for the enzymatic char-Menten constants of cytosolic cyclophilin (Cyp18) and acterization of PPIases is based on the acceleration of of the proteolytically sensitive FK506-binding proteinslow kinetic phases during the refolding of denatured like PPIase SlyD from Escherichia coli. Furthermore, proteins (4, 5). Obviously, the quasi-irreversible refoldsimilar inhibition constants were estimated for the reing of denatured proteins under strong native condiversible inhibition of human Cyp18 by cyclosporin A tions does not permit the measurement of enzymatic (CsA) with both the proteolytically coupled and the constants at the cis/trans equilibrium as well. novel uncoupled PPIase assay.