Abstract
Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.
Highlights
Our previous studies have dealt with the interactionbetween the two important physiological compounds, i.e. thevitamin C-bovine serumalbuminbinding behavior ( 5 ),the quenching of protein fluorescence induced by ascorbate (6) and oxidative modifications of carrier prochanged
We have investigated the action of a nonenzymatic metal-catalyzed oxidation (MCO)’ system
We have examined whether human serum albumin min (HSA) becomes more susceptible to proteolytic digestion following ascorbate modification, as reported for oxidatively inactivated enzymes (24).The results indicate that treated HSA does not show any change in its degradation by trypsin for 90 min at 37 "C and confirm that enhanced proteolysis requires stronger oxidative conditions
Summary
T = ( 1 - ( F / F ) x) 100 intervals, and the oxidative process was stopped by removing ascorbate by passage through a Sephadex G-25 column (Pharmacia) orby ultrafiltration through a Diaflo YMlO membrane (Amicon)A. scorbic acid wasmeasured in the eluate as absorbance at 265 nm In the latter procedure (employed for example in carbonyl groups determination and guanidine experiments) the first passawgerse performed with Tris plus mM DTPA. Fluorescence studies display a lowered (10-20%)quantum yield of oxidized proteins and a small blue-shift of the emission spectra (Fig. 2b). This sugpear more impaired than is with acrylamide. Difference betweenthe difference native HSA, respectively) is consistent with the higher hydrophobicity indicated by the slightly decreased solubility.Higher fluorescence spectrum relative to differentially excited native HSA affinity for thisnaturally occurring long-chain fatty acid (275 minus 295 nm excitation) and thraetlative to 72-h-treated HSA. Difference betweenthe difference native HSA, respectively) is consistent with the higher hydrophobicity indicated by the slightly decreased solubility.Higher fluorescence spectrum relative to differentially excited native HSA affinity for thisnaturally occurring long-chain fatty acid (275 minus 295 nm excitation) and thraetlative to 72-h-treated HSA. should be ascribed to anincreased number of exposed hydro-
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