Circular Dichroism Studies of Cyanate-Induced Conformational Changes in Hemoglobins A and S.
ABSTRACT Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying identical conformations about the heme groups of the respective proteins. The spectra of the oxygenated forms change little upon addition of KCNO, which is known to carbamoylate the NH2 terminals of the individual chains (Cerami and Manning, 1971). The spectra of the deoxygenated forms, on the other hand, are markedly altered. The decreased magnitude of the 430-nm extremum with increased cyanate concentration can be used to calculate an addition curve which becomes asymptotic at a cyanate:heme molar ratio of approximately 10(3). This conformational change occurs in the absence of O2 and has been predicted (Njikam et al.,1973); it can also be demonstrated by difference spectroscopy techniques, whereby a comparable addition curve can be constructed from changes in the 555-nm absorption, while the 541-nm absorption remains invariant. The change described corresponds to the formation of a new conformation, corresponding to carbamoyldeoxyhemoglobin, carrying one carbamoyl group per chain. In the presence of a small quantity of oxygen, however, the above reported changes in CD are accompanied by a concomitant rise in the 415-nm peak-corresponding to the formation of oxyhemoglobin-while those in the difference spectra reflect not only a change in the 555-nm band but also a parallel one at 541 nm, confirming the formation of oxyhemoglobin. Thus the conformation achieved upon carbamoylation of deoxyhemoglobin has the higher oxygen affinity predicted by Nigen et al. (1974) for carbamoyldeoxyhemoglobin. Cyanate has been used (Cerami and Manning, 1971) as an antisickling reagent in vivo and in vitro, but, although it has been shown that it binds covalently to the NH2-terminal residues of hemoglobin (Lee and Manning, 1973), its effect on hemoglobin conformation has not been previously shown nor has its mechanism of action been fully clarified. The results presented here show that the effect of cyanate on hemoglobin is the formation of a new conformation with heightened oxygen affinity. Since oxyHbS does not aggregate while deoxyHbS does, in a temperature-dependent fashion, the formation of carbamoyldeoxyHbS interferes with such aggregation in vitro in deoxygenated samples. In vivo, where there are generally low residual concentrations of O2, the formation of oxyHb is favored by the higher O2 affinity of carbamoyldeoxyHbS, and aggregation with concomitant red cell sickling is therefore disfavored.
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ABSTRACT: The conformational changes upon anaerobic carbamyl phosphate addition to dilute hemoglobin solutions have been studied by means of circular dichroism. Freshly prepared carbamyl phosphate stabilizes the conformation of pure deoxyhemoglobin without detectable NH2-terminal carbamylation. Addition of preincubated (i.e. partially converted to cyanate) carbamyl phosphate, however, results in such carbamylation and in the formation of the conformationally different carbamyldeoxyhemoglobin which exhibits enhanced oxygen affinity. Fresh carbamyl phosphate carbamylates hemoglobin in deoxygenated hemolysates containing erythrocyte phosphatases. Therefore the reversal of SS erythrocyte sickling by carbamyl phosphate is attributable to carbamylation by the carbamyl phosphate hydrolysis product, cyanate.Biochimica et Biophysica Acta 11/1977; 494(2):408-15. DOI:10.1016/0005-2795(77)90170-2 · 4.66 Impact Factor
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ABSTRACT: This study was initiated to characterize nonadrenergic-noncholinergic (NANC) inhibitory neurotransmission in penile corpus cavernosum. Using organ baths, isometric tension measurements were made in strips of human and rabbit corpus cavernosum. In examining endothelium-mediated responses, cumulative additions of exogenous acetylcholine elicited dose-dependent relaxations which were significantly reduced or completely inhibited in tissues treated with NG-monomethyl L-arginine (L-NMMA; an inhibitor of nitric oxide synthesis), oxyhemoglobin (a nitric oxide scavenger), or methylene blue (a guanylate cyclase blocker). Tissues exposed to hypoxic conditions (PO2 = 5-10 mmHg) also did not respond to exogenous acetylcholine. Mechanical removal of the endothelium in human corporal strips or in situ treatment of rabbit corpora with detergent blocked the relaxation to acetylcholine. Transmural electrical stimulation of corporal tissue strips denuded of functional endothelium, in the presence of adrenergic blockade with bretylium and muscarinic receptor blockade with atropine, caused frequency-dependent relaxation. This neurogenic relaxation was reduced or prevented by L-NMMA, oxyhemoglobin, methylene blue, and hypoxia. The effects of L-NMMA were reversed by L-arginine and the effects of hypoxia were readily reversed by normoxic conditions. Authentic, exogenous nitric oxide relaxed corporal strips which were contracted with adrenergic agonists and this effect was significantly inhibited by oxyhemoglobin. It is concluded that (a) endothelium-mediated responses of corpus cavernosum smooth muscle are mediated by a diffusible nitric oxide-like substance; (b) NANC neurogenic inhibitory responses do not require functional endothelium, and (c) nitric oxide, or a closely related substance, may act as an inhibitory neurotransmitter in penile corpus cavernosum smooth muscle.Journal of Clinical Investigation 08/1991; 88(1):112-8. DOI:10.1172/JCI115266 · 13.77 Impact Factor
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ABSTRACT: Carbamylation by urea-derived cyanate is a posttranslational modification of proteins increasing during chronic renal insufficiency, which alters structural and functional properties of proteins and modifies their interactions with cells. We report here the major structural alterations of type I collagen induced by carbamylation. Biophysical methods revealed that carbamylated collagen retained its triple-helical structure, but that slight changes destabilized some regions within the triple helix and decreased its ability to polymerize into normal fibrils. These changes were associated with the incapacity of carbamylated collagen to stimulate polymorphonuclear neutrophil oxidative functions. This process involved their interaction with LFA-1 integrin, but no subsequent p(125)FAK phosphorylation. Carbamylation of collagen might alter interactions between collagen and inflammatory cells in vivo and interfere with the normal remodeling of extracellular matrix, thus participating in the pathophysiological processes occurring during renal insufficiency.Chemistry & Biology 03/2006; 13(2):149-59. DOI:10.1016/j.chembiol.2005.11.005 · 6.59 Impact Factor