INTERACTION OF ACRYLAMIDE WITH PROTEINS IN THE CONCENTRATION RANGE USED FOR FLUORESCENCE QUENCHING STUDIES

C-14 labelled acrylamide was synthesized and used in equilibrium dialy sis measurements to study the binding of acrylamide to the proteins: h uman serum albumin (HSA), ovalbumin and cod parvalbumin III. Our inten t was to determine whether binding takes place in the concentration ra nge that is used for the study of fluorescence quenching by acrylamide . In contrast to previously published reports, we found that all the p roteins investigated did bind acrylamide. The affinity of this interac tion, when interpreted in terms of multiple independent binding equili bria, was very low, K(ass) congruent-to 0.5-2 M-1; however, due to mul tiple binding sites, a considerable amount of acrylamide is to be foun d in the protein phase at concentrations used for quenching experiment s. The number of binding sites seems to vary with the protein. A pH 7. 0 the binding to the 69 kD HSA corresponds to 14 +/- 8 sites, the bind ing to the 45 kD ovalbumin corresponds to 40 +/- 25 sites, whereas for the 11 kD cod parvalbumin the binding corresponds to only a few sites . The binding is very sensitive to pH and to the presence of cosolvent s such as glycerol. At pH 5.2, close to the isoionic point, the number of binding sites for acrylamide on HSA increases to > 100. The very w eak binding justifies an alternative description of the phenomena as a distribution equilibrium between two phases. In such a model we see t hat the formal concentration of acrylamide in the protein volume is in some cases higher than in solution (K(eq) = 1.2 for HSA at pH 5.2). T hese findings suggest that any model describing the quenching of fluor escence in proteins by acrylamide has to account for the presence of t wo pools of acrylamide and consequently for the presence of multiple m odes of quenching.