INTERACTION OF ALKALINE-PHOSPHATASE WITH CYTOCHROME-C

Alkaline phosphatase (AP) a protein which exhibits long-lived phosphor escence lifetime and ferricytochrome c as a phosphorescence quenching agent were examined. The excitation of the tryptophan triplet state re sulted in cylochrorne c reduction confirming long-range electron trans fer as the quenching mechanism. The rate of electron transfer was not related to the length of the illumination interval; an additional reac tion between the two proteins leading to cytochrome c reduction was de tected. The reaction which proceeded in the dark; was not sensitive to oxygen, was dependent on pH, and on the AP to cytochrome c ratio. At optimum 68 +/- 4% of the total cytochrome c could be reduced due to th e presence of AP. On incubation of the two proteins the conformation o f cytochrome c was altered as was evidenced by its decreased reducibil ity by ascorbate, by the disappearance of the absorption band at 695 n m, by the appearance of the new band at 620-640 nm, and by a change in circular dichroism spectra witnessing a structural alteration in the vicinity of the heme cleft. This was characterized by a profound incre ase in positive elipticity at 400 nm and by a reversible change in the magnitude of negative elipticity at 417 nm. The reaction was not sign ificantly affected by the addition of sulfhydryl-binding and metal-com plexing agents.