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Amphoteric Behavior of Bovine Plasma Albumin and Its Detergent Complexes

From the ¹ From the Department of Chemistry, Purdue University, Lafayette, Indiana 47907

Hydrogen ion titration curves have been determined for two stoichiometric albumin-detergent complexes, AD₁₁ and AD₇₆, which are formed through interaction between bovine plasma albumin and the detergent sodium dodecyl benzenesulfonate. The amphoteric properties of the protein moiety in these two complexes have been compared with one another and with those of the uncombined protein. In this analysis, electrophoretic mobility data were used for making the necessary corrections for electrostatic interaction. Tyrosyl ionization curves were established for all three forms by measuring the change with pH of absorption at 295 mµ. The most significant differences found are in the tyrosyl ionization. For albumin itself, the apparent value of pK_int is approximately 11.0, and it is concluded that some masked tyrosyl residues are exposed in a cooperative structural change accompanying the ionization. In the case of AD₁₁, tyrosyl ionization is distinctly hypersharp and the apparent pK_int is 11.3, implying that in this form the protein is stabilized against unmasking of the tyrosyl residues. In AD₇₆ the apparent pK_int is again near 11.0. The titration behavior of imidazole groups is virtually identical in the native protein and in the AD₁₁ complex. In both cases it is necessary to assume two classes of imidazole groups in order to fit the titration curve. In AD₇₆ the titration of these groups can be fitted adequately with a single pK_int of 7.30. Surprisingly, very little difference is seen between the various forms in the titration of the -amino groups. This fact permits the conclusion that electrostatic interaction between the detergent anions and cationic ammonium groups cannot contribute more than 700 cal to the free energy of binding, whereas the total binding free energy (on the unitary scale) must be of the order of 10,000 cal. The fact that the stoichiometry of the complexes is virtually unaltered over the pH range 6.9 to 11.6, over which range the total number of cationic sites in the protein falls from approximately 100 to 40, supports the conclusion that binding is not primarily electrostatic but is probably due to hydrophobic interactions. To a first approximation, all results suggest that the protein in AD₁₁ is essentially in the native state and in fact is stabilized with respect to the structural alteration which must occur on conversion to AD₇₆.

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