SEC-HPLC and rapid kinetics of Hp binding to Hb and PolyHb. (A) SEC-HPLC chromatograms of Hb and PolyHb with and without Hp. The absorbance was monitored at 413 nm to detect heme. The peak for unmodified Hb elutes at ∼9.6 minutes. Each chromatogram was normalized to the peak area under the curve before the addition of Hp. The molar ratio of Hb to Hp was 1.5:1; the molar ratio of PolyHb to Hp was 1:2. (B) The percent composition based on the approximate size order was determined with a Gaussian deconvolution of the resulting chromatograms. (C) Time courses of Hp (0.25 μM, Hb tetramer binding basis) and Hb/PolyHb (on an Hb tetramer molar basis) were fit to monoexponential equations (dashed lines). Experimental data show an average of 10 kinetic traces. The reactions were monitored by the fluorescence emission using a 310 nm high-pass filter at 20°C. Phosphate-buffered saline (0.1 M, pH 7.4) was used as the reaction buffer. (D) Second-order rate constants of Hp binding to Hb/PolyHb derived as a function of Hb concentration on a Hb tetramer molar basis.