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. 1992 Oct 1;287(Pt 1):107–112. doi: 10.1042/bj2870107

Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.

S A Santini 1, A Mordente 1, E Meucci 1, G A Miggiano 1, G E Martorana 1
PMCID: PMC1133130  PMID: 1417762

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amici A., Levine R. L., Tsai L., Stadtman E. R. Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions. J Biol Chem. 1989 Feb 25;264(6):3341–3346. [PubMed] [Google Scholar]
  2. Berger J. W., Vanderkooi J. M. Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy. Biochemistry. 1989 Jun 27;28(13):5501–5508. doi: 10.1021/bi00439a027. [DOI] [PubMed] [Google Scholar]
  3. Bloemendal H. Lens proteins. CRC Crit Rev Biochem. 1982;12(1):1–38. doi: 10.3109/10409238209105849. [DOI] [PubMed] [Google Scholar]
  4. Bloemendal H. The vertebrate eye lens. Science. 1977 Jul 8;197(4299):127–138. doi: 10.1126/science.877544. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Castagnola M., Cassiano L., De Cristofaro R., Landolfi R., Rossetti D. V., Bettolo G. B. High-performance liquid chromatography in protein sequence determinations. J Chromatogr. 1988 May 25;440:231–251. doi: 10.1016/s0021-9673(00)94527-8. [DOI] [PubMed] [Google Scholar]
  7. Davies K. J., Delsignore M. E., Lin S. W. Protein damage and degradation by oxygen radicals. II. Modification of amino acids. J Biol Chem. 1987 Jul 15;262(20):9902–9907. [PubMed] [Google Scholar]
  8. Davies K. J., Delsignore M. E. Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure. J Biol Chem. 1987 Jul 15;262(20):9908–9913. [PubMed] [Google Scholar]
  9. Dunn J. A., Ahmed M. U., Murtiashaw M. H., Richardson J. M., Walla M. D., Thorpe S. R., Baynes J. W. Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate. Biochemistry. 1990 Dec 11;29(49):10964–10970. doi: 10.1021/bi00501a014. [DOI] [PubMed] [Google Scholar]
  10. Eftink M. R., Ghiron C. A. Exposure of tryptophanyl residues and protein dynamics. Biochemistry. 1977 Dec 13;16(25):5546–5551. doi: 10.1021/bi00644a024. [DOI] [PubMed] [Google Scholar]
  11. Garland D. Role of site-specific, metal-catalyzed oxidation in lens aging and cataract: a hypothesis. Exp Eye Res. 1990 Jun;50(6):677–682. doi: 10.1016/0014-4835(90)90113-9. [DOI] [PubMed] [Google Scholar]
  12. Garland D., Russell P., Zigler J. S., Jr The oxidative modification of lens proteins. Basic Life Sci. 1988;49:347–352. doi: 10.1007/978-1-4684-5568-7_52. [DOI] [PubMed] [Google Scholar]
  13. Garland D., Zigler J. S., Jr, Kinoshita J. Structural changes in bovine lens crystallins induced by ascorbate, metal, and oxygen. Arch Biochem Biophys. 1986 Dec;251(2):771–776. doi: 10.1016/0003-9861(86)90389-9. [DOI] [PubMed] [Google Scholar]
  14. Hand S. C., Somero G. N. Urea and methylamine effects on rabbit muscle phosphofructokinase. Catalytic stability and aggregation state as a function of pH and temperature. J Biol Chem. 1982 Jan 25;257(2):734–741. [PubMed] [Google Scholar]
  15. Hollecker M., Creighton T. E. Effect on protein stability of reversing the charge on amino groups. Biochim Biophys Acta. 1982 Mar 4;701(3):395–404. doi: 10.1016/0167-4838(82)90243-6. [DOI] [PubMed] [Google Scholar]
  16. Hunt J. V., Dean R. T., Wolff S. P. Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochem J. 1988 Nov 15;256(1):205–212. doi: 10.1042/bj2560205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ingolia T. D., Craig E. A. Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2360–2364. doi: 10.1073/pnas.79.7.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kramps J. A., de Man B. M., de Jong W. W. The primary structure of the B2 chain of human alpha-crystallin. FEBS Lett. 1977 Feb 15;74(1):82–84. doi: 10.1016/0014-5793(77)80757-6. [DOI] [PubMed] [Google Scholar]
  19. Kuwajima K., Nitta K., Yoneyama M., Sugai S. Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride. J Mol Biol. 1976 Sep 15;106(2):359–373. doi: 10.1016/0022-2836(76)90091-7. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lehrer S. S. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry. 1971 Aug 17;10(17):3254–3263. doi: 10.1021/bi00793a015. [DOI] [PubMed] [Google Scholar]
  22. Lenz A. G., Costabel U., Shaltiel S., Levine R. L. Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride. Anal Biochem. 1989 Mar;177(2):419–425. doi: 10.1016/0003-2697(89)90077-8. [DOI] [PubMed] [Google Scholar]
  23. Levine R. L. Mixed-function oxidation of histidine residues. Methods Enzymol. 1984;107:370–376. doi: 10.1016/0076-6879(84)07025-7. [DOI] [PubMed] [Google Scholar]
  24. Liang J. N., Bose S. K., Chakrabarti B. Age-related changes in protein conformation in bovine lens crystallins. Exp Eye Res. 1985 Mar;40(3):461–469. doi: 10.1016/0014-4835(85)90159-9. [DOI] [PubMed] [Google Scholar]
  25. Liang J. N., Chakrabarti B. Spectroscopic investigations of bovine lens crystallins. 1. Circular dichroism and intrinsic fluorescence. Biochemistry. 1982 Apr 13;21(8):1847–1852. doi: 10.1021/bi00537a022. [DOI] [PubMed] [Google Scholar]
  26. Liang J. N., Pelletier M. R. Destabilization of lens protein conformation by glutathione mixed disulfide. Exp Eye Res. 1988 Jul;47(1):17–25. doi: 10.1016/0014-4835(88)90020-6. [DOI] [PubMed] [Google Scholar]
  27. Maiti M., Kono M., Chakrabarti B. Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin. FEBS Lett. 1988 Aug 15;236(1):109–114. doi: 10.1016/0014-5793(88)80295-3. [DOI] [PubMed] [Google Scholar]
  28. Meucci E., Mordente A., Martorana G. E. Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging. J Biol Chem. 1991 Mar 15;266(8):4692–4699. [PubMed] [Google Scholar]
  29. Miggiano G. A., Mordente A., Pischiutta M. G., Martorana G. E., Castelli A. Early conformational changes and activity modulation induced by guanidinium chloride on intestinal alkaline phosphatase. Biochem J. 1987 Dec 1;248(2):551–556. doi: 10.1042/bj2480551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mordente A., Martorana G. E., Miggiano G. A., Meucci E., Santini S. A., Castelli A. Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase. Arch Biochem Biophys. 1988 Aug 1;264(2):502–509. doi: 10.1016/0003-9861(88)90315-3. [DOI] [PubMed] [Google Scholar]
  31. Mordente A., Miggiano G. A., Martorana G. E., Meucci E., Santini S. A., Castelli A. Alkaline phosphatase inactivation by mixed function oxidation systems. Arch Biochem Biophys. 1987 Oct;258(1):176–185. doi: 10.1016/0003-9861(87)90334-1. [DOI] [PubMed] [Google Scholar]
  32. Oliver C. N., Levine R. L., Stadtman E. R. A role of mixed-function oxidation reactions in the accumulation of altered enzyme forms during aging. J Am Geriatr Soc. 1987 Oct;35(10):947–956. doi: 10.1111/j.1532-5415.1987.tb02297.x. [DOI] [PubMed] [Google Scholar]
  33. Ortwerth B. J., Olesen P. R. Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction. Biochim Biophys Acta. 1988 Aug 31;956(1):10–22. doi: 10.1016/0167-4838(88)90292-0. [DOI] [PubMed] [Google Scholar]
  34. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  35. Pace C. N. The stability of globular proteins. CRC Crit Rev Biochem. 1975 May;3(1):1–43. doi: 10.3109/10409237509102551. [DOI] [PubMed] [Google Scholar]
  36. Phillips S. R., Wilson L. J., Borkman R. F. Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins. Curr Eye Res. 1986 Aug;5(8):611–619. doi: 10.3109/02713688609015126. [DOI] [PubMed] [Google Scholar]
  37. Prinsze C., Dubbelman T. M., Van Steveninck J. Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals. Biochim Biophys Acta. 1990 Apr 19;1038(2):152–157. doi: 10.1016/0167-4838(90)90198-o. [DOI] [PubMed] [Google Scholar]
  38. Rao G. N., Lardis M. P., Cotlier E. Acetylation of lens crystallins: a possible mechanism by which aspirin could prevent cataract formation. Biochem Biophys Res Commun. 1985 May 16;128(3):1125–1132. doi: 10.1016/0006-291x(85)91057-5. [DOI] [PubMed] [Google Scholar]
  39. Rivett A. J., Levine R. L. Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes. Arch Biochem Biophys. 1990 Apr;278(1):26–34. doi: 10.1016/0003-9861(90)90226-o. [DOI] [PubMed] [Google Scholar]
  40. Rivett A. J. Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases. J Biol Chem. 1985 Jan 10;260(1):300–305. [PubMed] [Google Scholar]
  41. Rivett A. J. Regulation of intracellular protein turnover: covalent modification as a mechanism of marking proteins for degradation. Curr Top Cell Regul. 1986;28:291–337. doi: 10.1016/b978-0-12-152828-7.50010-x. [DOI] [PubMed] [Google Scholar]
  42. Rivett A. J., Roseman J. E., Oliver C. N., Levine R. L., Stadtman E. R. Covalent modification of proteins by mixed-function oxidation: recognition by intracellular proteases. Prog Clin Biol Res. 1985;180:317–328. [PubMed] [Google Scholar]
  43. Sen A. C., Chakrabarti B. Effect of acetylation by aspirin on the thermodynamic stability of lens crystallins. Exp Eye Res. 1990 Dec;51(6):701–709. doi: 10.1016/0014-4835(90)90055-y. [DOI] [PubMed] [Google Scholar]
  44. Siezen R. J., Bindels J. G., Hoenders H. J. The quaternary structure of bovine alpha-crystallin. Effects of variation in alkaline pH, ionic strength, temperature and calcium ion concentration. Eur J Biochem. 1980 Oct;111(2):435–444. doi: 10.1111/j.1432-1033.1980.tb04958.x. [DOI] [PubMed] [Google Scholar]
  45. Siezen R. J., Bindels J. G. Stepwise dissociation/denaturation and reassociation/renaturation of bovine alpha-crystallin in urea and guanidine hydrochloride: sedimentation, fluorescence, near-ultraviolet and far-ultraviolet circular dichroism studies. Exp Eye Res. 1982 Jun;34(6):969–983. doi: 10.1016/0014-4835(82)90077-x. [DOI] [PubMed] [Google Scholar]
  46. Siezen R. J., Owen E. A., Kubota Y., Ooi T. Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles. Biochim Biophys Acta. 1983 Oct 17;748(1):48–55. doi: 10.1016/0167-4838(83)90026-2. [DOI] [PubMed] [Google Scholar]
  47. Siezen R. J. Reflections on the internal primary, secondary and tertiary structure homology of the eye lens proteins alpha-, beta- and gamma-crystallin. FEBS Lett. 1981 Oct 12;133(1):1–8. doi: 10.1016/0014-5793(81)80459-0. [DOI] [PubMed] [Google Scholar]
  48. Slight S. H., Feather M. S., Ortwerth B. J. Glycation of lens proteins by the oxidation products of ascorbic acid. Biochim Biophys Acta. 1990 May 8;1038(3):367–374. doi: 10.1016/0167-4838(90)90250-j. [DOI] [PubMed] [Google Scholar]
  49. Stadtman E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic Biol Med. 1990;9(4):315–325. doi: 10.1016/0891-5849(90)90006-5. [DOI] [PubMed] [Google Scholar]
  50. Steadman B. L., Trautman P. A., Lawson E. Q., Raymond M. J., Mood D. A., Thomson J. A., Middaugh C. R. A differential scanning calorimetric study of the bovine lens crystallins. Biochemistry. 1989 Dec 12;28(25):9653–9658. doi: 10.1021/bi00451a017. [DOI] [PubMed] [Google Scholar]
  51. Steinberg I. Z. Long-range nonradiative transfer of electronic excitation energy in proteins and polypeptides. Annu Rev Biochem. 1971;40:83–114. doi: 10.1146/annurev.bi.40.070171.000503. [DOI] [PubMed] [Google Scholar]
  52. Stevens V. J., Rouzer C. A., Monnier V. M., Cerami A. Diabetic cataract formation: potential role of glycosylation of lens crystallins. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2918–2922. doi: 10.1073/pnas.75.6.2918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  54. Thomson J. A., Augusteyn R. C. On the structure of alpha m-crystallin. The reversibility of urea dissociation. J Biol Chem. 1984 Apr 10;259(7):4339–4345. [PubMed] [Google Scholar]
  55. Varma S. D., Richards R. D. Ascorbic acid and the eye lens. Ophthalmic Res. 1988;20(3):164–173. doi: 10.1159/000266579. [DOI] [PubMed] [Google Scholar]
  56. Wistow G. Domain structure and evolution in alpha-crystallins and small heat-shock proteins. FEBS Lett. 1985 Feb 11;181(1):1–6. doi: 10.1016/0014-5793(85)81102-9. [DOI] [PubMed] [Google Scholar]
  57. Wolff S. P., Jiang Z. Y., Hunt J. V. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Radic Biol Med. 1991;10(5):339–352. doi: 10.1016/0891-5849(91)90040-a. [DOI] [PubMed] [Google Scholar]
  58. Wong K. P., Tanford C. Denaturation of bovine carbonic anhydrase B by guanidine hydrochloride. A process involving separable sequential conformational transitions. J Biol Chem. 1973 Dec 25;248(24):8518–8523. [PubMed] [Google Scholar]
  59. de Jong W. W., Terwindt E. C., Bloemendal H. The amino acid sequence of the A chain of human alpha-crystallin. FEBS Lett. 1975 Oct 15;58(1):310–313. doi: 10.1016/0014-5793(75)80286-9. [DOI] [PubMed] [Google Scholar]
  60. van den Oetelaar P. J., Hoenders H. J. Folding-unfolding and aggregation-dissociation of bovine alpha-crystallin subunits; evidence for unfolding intermediates of the alpha A subunits. Biochim Biophys Acta. 1989 Mar 16;995(1):91–96. doi: 10.1016/0167-4838(89)90238-0. [DOI] [PubMed] [Google Scholar]

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