Resolution of binding sites in β-Lactoglobulin

Rex Lovrien*, Wayne Anderson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

β-Lactoglobulin has been considered to have two or three strong binding sites for organic anions. By means of calorimetry with a very strongly binding anion, dodecyl-sulfate, enthalpic titration indicates two binding sites available on the acid side of the pH 7.5 conformation transition. There remain two sites available on the alkaline side of the same transition. One of these sites was resolved as a single, rather strongly binding site by means of spectrofluorimetry of a fluorescent organic anion at concentrations of the order of 10-5 m. The second site is a considerably weaker anionbinding site, but was not resolved as such. Instead, dialysis equilibrium-binding experiments using the same anion, at concentrations of the order of 10-4 m were performed. The data, used in Scatchard's method of plotting, yield the average of the two binding sites at this higher organic anion concentration. The molar association constants (which may be corrected further for statistical and electrostatic factors) for β-lactoglobulin at pH 8 with 0.02 m Cl- and a N-methylanilinonaphthalene sulfonate anion, are K1 = 3.4 ± 0.5 × 105 for n1 = 0.95 ± 0.10 sites, fully resolved, and K2 = 2.0 ± 0.2 × 104 for n2 = 2.0 ±0.2 sites which include the first strong binding site and the second weaker site. The K2 cited refers to an average binding constant for both sites. Probably Cl- competition for the same anion-binding sites, at this pH, is minimal. By using fluorescent compounds and adequate control of excitation wavelength, it is probable that binding constants for individual strong binding sites can be worked out for fluorescent compound binding to a variety of other proteins.

Original languageEnglish (US)
Pages (from-to)139-144
Number of pages6
JournalArchives of biochemistry and biophysics
Volume131
Issue number1
DOIs
StatePublished - Apr 1969

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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