The effect of complex-formation with polyanions on the redox properties of cytochrome c

Abstract

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.

The oxidative mechanism of heparin interferes with radical production by glucose and reduces the degree of glycooxidative modifications on human serum albumin

“…It is possible that heparin binding can either quench radicals formed by glucose or even prevent their formation by interfering with glucose binding. Heparin binding to HSA may also change the reaction potential of the protein with glucose, similar to that observed with heparin-cytochrome c complexes, which display a modified reactivity with other redox agents [48]. Whatever the mechanism, our results are in keeping with numerous reports in the literature stressing both the antioxidative properties of heparin [49] and its capacity to enhance the oxidative modifications of protein [50] including a higher susceptibility to proteolytic digestion.…”

Electrostatic interactions in the heparin-enhanced reaction between human thrombin and antithrombin

“…The binding curve (partition coeffi-cient plotted against heparin concentration) (Fig. 1) is more complex than can be accounted for by means of the single-site models that have previously been applied for a quantitative determination of binding constants in more simple cases of macromolecular electrostatic interaction (see, e.g., Petersen, 1978; Petersen & Cox, 1980). This is apparent from the biphasic character -of the binding curve, which is most pronounced in the LiCl-containing two-phase system.…”

Protein surface recognition by synthetic agents: Design and structural requirements of a family of artificial receptors that bind to cytochrome C

“…If 9a were binding to this region, then it could potentially block the approach of oxidizing or reducing agents to cytochrome c. To test this we have investigated the effect of 9a on the interaction of Fe(III) cytochrome c with ascorbic acid as reducing agent in phosphate buffer. 19,20 In the absence of receptor ). Mochan and Nicholls had previously reported 19 that cytochrome c peroxidase caused a very similar inhibition of ascorbate reduction through complex formation to the heme edge region of cytochrome c (for a crystal structure of the complex between cytochrome c and cytochrome c peroxidase, see Ref.…”