Identification of the binding site for plasma prekallikrein in human high molecular weight kininogen. A region from residues 185 to 224 of the kininogen light chain retains full binding activity.
We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.
D001665:Binding Sites; D003488:Cyanogen Bromide; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D007610:Kallikreins / Q000502:physiology*; D007704:Kininogens / Q000097:blood*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000097:blood; D011288:Prekallikrein / Q000502:physiology*; D013861:Thiocyanates; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007704:Kininogens; D010446:Peptide Fragments; D013861:Thiocyanates; D011288:Prekallikrein; D007610:Kallikreins; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; D003488:Cyanogen Bromide