Interaction of alkaline phosphatase with cytochrome c.
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.
Dadák
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V
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. dadak@chemi.muni.cz
Vrána
O
O
Nováková
O
O
Antalík
M
M
D000332:Aerobiosis; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000693:Anaerobiosis; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D003624:Darkness; D004492:Edetic Acid / Q000494:pharmacology; D004579:Electron Transport / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008027:Light; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D014364:Tryptophan
D002614:Chelating Agents; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D011189:Potassium Chloride; D014364:Tryptophan; D004492:Edetic Acid; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid