Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.
Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.
Santini
S A
SA
Istituto di Chimica Biologica, Università Cattolica del S. Cuore, Facoltà di Medicina e Chirurgia, Agostino Gemelli, Roma, Italy.
Mordente
A
A
Meucci
E
E
Miggiano
G A
GA
Martorana
G E
GE
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D003459:Crystallins / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007454:Iodides / Q000737:chemistry; D008027:Light; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D000178:Acrylamides; D003459:Crystallins; D006146:Guanidines; D007454:Iodides; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D019791:Guanidine; D001205:Ascorbic Acid