The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.
NMR spectroscopy is an important technique for the study of biological fluids and intact cells (1-8), Compared with other analytical methods, it is nondestructive and noninvasive, and it allows delicately balanced chemical and cellular processes to be observed directly at the molecular level. 13C, 15N, 31P, and !H NMR spectroscopies have all been used to study biological fluids and/or intact cells.Because of the inherent low NMR sensitivities and low natural abundances of 13C and 15N, isotopically enriched compounds generally are used in 13C and l5N NMR studies. The advantage is a relatively simple spectrum that consists of resonances from the enriched compounds and their metabolites superimposed on much weaker background resonances. However, isotopically enriched compounds are required, and, in the case of intact cell studies, they must be incorporated into the cells. Nevertheless, 13C and 15N NMR with isotopically enriched compounds are widely used and important methods for the study of metabolic processes in intact cells, 31P, which has a natural abundance of 100% and a high inherent NMR sensitivity, is also widely used. Because there are few phosphorus-containing compounds at detectable levels, 31P NMR spectra of biological fluids and intact cells generally are relatively simple. For the same reason, however, 31P NMR can be used to study only a limited number of compounds.the analog-to-digital converter. This spectrum and all the other spectra presented In this article were measured with a Varlan VXR-500S spectrometer operated in the unlocked mode.
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