Interaction of alkaline phosphatase with cytochrome c

Abstract

Alkaline phosphatase (AP) a protein which exhibits long-lived phosphorescence lifetime and ferricytochrome c as a phosphorescence quenching agent were examined. The excitation of the tryptophan triplet state resulted in cytochrome c reduction confirming long-range electron transfer as the quenching mechanism. The rate of electron transfer was not related to the length of the illumination interval; an additional reaction between the two proteins leading to cytochrome c reduction was detected. 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 the presence of AP. On incubation of the two proteins the conformation of cytochrome c was altered as was evidenced by its decreased reducibility by ascorbate, by the disappearance of the absorption band at 695 nm, 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 increase in positive elipticity at 400 nm and by a reversible change in the magnitude of negative elipticity at 417 nm. The reaction was not significantly affected by the addition of sulfhydryl-binding and metal-complexing agents.