A NEW METHOD FOR DETERMINING THE DISSOCIATION-CONSTANT BETWEEN MACROMOLECULES BY GEL-PERMEATION

A method was devised for determining the Kd of binding of heparin to protein. The method was based on gel permeation chromatography with the eluting buffer containing a fluorescent macromolecule. Fluorescence intensity change, Qobs, induced by another macromolecule, was monitored using a flow cell. The maximal change at full saturation, Qmax, was determined from a double-reciprocal plot of Qobs and the concentration of the other macromolecule. The Kd, was calculated from an equation involving Qobs, Qmax, and the given concentration of the other macromolecule [E0]. The Kd between bovine serum albumin and N-fluoresceinylthiocarbamoyl-heparin was calculated to be 7.3 .+-. 0.52 .times. 10-5 M by this method. The Kd between the protein and unlabeled heparin was 3.5 .times. 10-5 M by spectrophotometric titration and 3.6 .times. 10-5 M by measurement of inhibition of metachromasia. The present method is available for direct determination of the Kd values of various macromolecules with different fluorescence characteristics.