Antithrombin III and its interaction with heparin. Comparison of the human, bovine, and porcine proteins by 1H NMR spectroscopy.
1H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their 1H NMR spectra. In addition, aromatic and methyl proton resonances in upfield-shifted positions appear to be common to all three proteins and suggest similar tertiary structures. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pKa's are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The 1H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. These resonances include those of histidine C(2) and C(4) protons, of 10-20 other aromatic protons, of a methyl group, and also of protons with chemical shifts similar to those of lysine and/or arginine side chains. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D002417:Cattle; D006493:Heparin / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine
D000596:Amino Acids; D009844:Oligosaccharides; D006639:Histidine; D000990:Antithrombin III; D006493:Heparin