Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Mar 1;282(Pt 2):589–593. doi: 10.1042/bj2820589

Spectrofluorimetric assessment of the surface hydrophobicity of proteins.

M Cardamone 1, N K Puri 1
PMCID: PMC1130822  PMID: 1546973

Abstract

The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution.

Full text

PDF
589

Selected References

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

  1. Bigelow C. C. On the average hydrophobicity of proteins and the relation between it and protein structure. J Theor Biol. 1967 Aug;16(2):187–211. doi: 10.1016/0022-5193(67)90004-5. [DOI] [PubMed] [Google Scholar]
  2. Birdsall B., King R. W., Wheeler M. R., Lewis C. A., Jr, Goode S. R., Dunlap R. B., Roberts G. C. Correction for light absorption in fluorescence studies of protein-ligand interactions. Anal Biochem. 1983 Jul 15;132(2):353–361. doi: 10.1016/0003-2697(83)90020-9. [DOI] [PubMed] [Google Scholar]
  3. Davies R. C., Neuberger A., Wilson B. M. The dependence of lysozyme activity on pH and ionic strength. Biochim Biophys Acta. 1969 Apr 22;178(2):294–305. doi: 10.1016/0005-2744(69)90397-0. [DOI] [PubMed] [Google Scholar]
  4. Fausnaugh J. L., Kennedy L. A., Regnier F. E. Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins. J Chromatogr. 1984 Dec 28;317:141–155. doi: 10.1016/s0021-9673(01)91654-1. [DOI] [PubMed] [Google Scholar]
  5. Joniau M., Bloemmen J., Lontie R. The reaction of 4-(p-sulfophenylazo)-2-mercuriphenol with the thiol groups of proteins. Biochim Biophys Acta. 1970 Sep 29;214(3):468–477. doi: 10.1016/0005-2795(70)90306-5. [DOI] [PubMed] [Google Scholar]
  6. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  7. Kennedy R. M. Hydrophobic chromatography. Methods Enzymol. 1990;182:339–343. doi: 10.1016/0076-6879(90)82029-2. [DOI] [PubMed] [Google Scholar]
  8. Klotz I. M., Hunston D. L. Properties of graphical representations of multiple classes of binding sites. Biochemistry. 1971 Aug 3;10(16):3065–3069. doi: 10.1021/bi00792a013. [DOI] [PubMed] [Google Scholar]
  9. Murphy K. P., Privalov P. L., Gill S. J. Common features of protein unfolding and dissolution of hydrophobic compounds. Science. 1990 Feb 2;247(4942):559–561. doi: 10.1126/science.2300815. [DOI] [PubMed] [Google Scholar]
  10. Nozaki Y., Tanford C. The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. J Biol Chem. 1971 Apr 10;246(7):2211–2217. [PubMed] [Google Scholar]
  11. Schein C. H. Solubility as a function of protein structure and solvent components. Biotechnology (N Y) 1990 Apr;8(4):308–317. doi: 10.1038/nbt0490-308. [DOI] [PubMed] [Google Scholar]
  12. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  13. TABORSKY G. Inactivation of ribonuclease by phosphorylation. J Biol Chem. 1959 Nov;234:2915–2920. [PubMed] [Google Scholar]
  14. Thakur A. K., Jaffe M. L., Rodbard D. Graphical analysis of ligand-binding systems: evaluation by Monte Carlo studies. Anal Biochem. 1980 Sep 15;107(2):279–295. doi: 10.1016/0003-2697(80)90385-1. [DOI] [PubMed] [Google Scholar]
  15. WEBER G., LAURENCE D. J. Fluorescent indicators of adsorption in aqueous solution and on the solid phase. Biochem J. 1954 Jan 16;56(325TH):xxxi–xxxi. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES