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The pKa values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by 1H NMR spectroscopy. The 1H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lower pKa value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. The pKa values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The 1H NMR resonance of the histidine of human platelet factor 4 exhibiting the lower pKa value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higher pKa, thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.