ascorbic-acid and alpha-chymotrypsin

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.

Topics: 2,2'-Dipyridyl; Apoptosis; Ascorbic Acid; Binding Sites; Cell Line, Tumor; Cell Membrane Permeability; Chymotrypsin; Coordination Complexes; Cytochromes c; Humans; Kinetics; Models, Molecular; Oxidation-Reduction; Potentiometry; Protein Binding; Ruthenium; Thermodynamics

Protein radicals were selectively generated by reaction with azide radicals on Trp and Tyr residues in insulin, beta-lactoglobulin, pepsin, chymotrypsin, and bovine serum albumin at rate constants in the range (2.9-19)x10(8) M(-1) s(-1). Monohydrogen ascorbate reduced tryptophanyl radicals in chymotrypsin and pepsin with rate constants in the narrow range of (1.6-1.8)x10(8) M(-1) s(-1), whereas beta-lactoglobulin tryptophanyl radicals reacted almost 10 times slower. The corresponding values for the protein tyrosyl radicals were about an order of magnitude smaller. Comparison of the rate constants of reactions of free and protein-bound tryptophanyl and tyrosyl radicals showed that, in most cases, the location of the radicals in the protein chain did not constitute a major barrier to the reaction with monohydrogen ascorbate. The results suggest that, under physiological concentrations of dioxygen, monohydrogen ascorbate is likely to be a significant target of protein radicals. It seems likely, therefore, that reaction with protein radicals may be responsible for much of the well-documented loss of ascorbate in living organisms subjected to oxidative stress.

Topics: Animals; Ascorbic Acid; Cattle; Chymotrypsin; In Vitro Techniques; Insulin; Lactoglobulins; Oxidative Stress; Pepsin A; Protein Binding; Protein Processing, Post-Translational; Pulse Radiolysis; Reactive Nitrogen Species; Reactive Oxygen Species; Serum Albumin, Bovine; Swine; Tryptophan; Tyrosine