Dye binding by protein as applied to quantitative paper electrophoresis

1. (1) The binding of acid dyes by denatured protein on filter paper has been investigated with special reference to Light Green. 2. (2) The uptake of dye, measured by elution, has been shown to vary according to the method of denaturation, the nature of the dye solvent (particularly the salt and and ethanol concentration) and finally with the duration and temperature of the subsequent washing procedure. 3. (3) Albumin and globulin do not behave in the same manner with different denaturing agents so that different methods give different albumin/globulin dye uptake ratios. 4. (4) The stoichiometric relationship between dye and protein is consistently nonlinear even at lower concentrations of protein on the paper and the same pattern can be demonstrated with protein on cellulose acetate membrane. It is suggested that this non-linear relationship at low protein densities is due to interference by the cellulose supporting medium and other anions. 5. (5) Intrinsic constants and the number of binding sites at infinite dye concentration have been measured under standardized conditions for Light Green, Brom-Cresol Green and Lissamine Green. The results have been compared and differences discussed. 6. (6) In the quantitative measurement of protein based on dye uptake, the importance of restricting the protein density on the paper is stressed.