COMPARATIVE BINDING-STUDIES OF CYCLOPHILINS TO CYCLOSPORINE-A AND DERIVATIVES BY FLUORESCENCE MEASUREMENTS

The interaction of cyclosporin A and cyclosporin derivatives with cyclophilins A, B, and C has been investigated by means of fluorescence measurement techniques. Since Trp-121 of cyclophilin A is in close contact with bound cyclosporins and changes its fluorescence emission upon binding, direct estimation of K-d values for cyclosporins is straightforward in this case. Cyclophilins B and C, however, display no evident binding-dependent fluorescence changes suitable for the estimation of their binding affinities. This problem can be circumvented by measuring the variations of fluorescence emission intensities of a mixture of cyclophilin A and the fluorescence measurements unsuitable for cyclosporin binder as a function of ligand concentration. Application of a mixed-mode kinetic analysis then allows the calculation of the cyclosporin binding affinity of the second binder in the system. The dissociation constant for cyclosporin A/cyclophilin A was found to be 36.8 nM. Mixed-mode kinetic calculations yielded K-d values of 9.8 and 90.8 nM for cyclophilins B and C, respectively. The analysis was extended to noncyclophilin (weak) cyclosporin binders such as calmodulin and actin, resulting in approximate K-d values of 1.2 and 5.7 mu M, respectively. Using the same approach, the K-d values of a series of different cyclosporin derivatives were determined. (C) 1994 Academic Press, Inc.