Influência de combinações de diferentes caotrópicos no espectro visível da hemoglobina e na estabilidade de membrana de eritrócitos humanos

Abstract

CHAPTER II: This study aimed to investigate the influence of combinations of urea, ethanol and hypotonicity on the stability and spectrum of human hemoglobin. Protocols with fixed incubation time of 30 minutes at 37 °C were used to analyze the spectra of commercial hemoglobin and hemoglobin of venous blood obtained from 20 healthy volunteers. The visible spectrum of hemoglobin obtained in our laboratory was consistent with that of hemoglobin and its stability was monitored at 540 nm, while the spectrum of commercial hemoglobin was consistent with that of metahemoglobin and its stability was monitored at 630 nm. Concentrations of 0-5 M urea, 0-20% (V/V) ethanol in the absence and presence of 1.5 M urea and 0-1 g/dL NaCl in the absence and presence of 1 M urea produced discrete changes in the visible spectrum of hemoglobin. However, beyond 5 M urea and 20% ethanol, oxyhemoglobin underwent spectral changes probably associated with the formation of desoxyhemoglobin and then its denaturation. Metahemoglobin showed greater vulnerability to the effects of chaotropic studied. As the transitions of hemolysis of erythrocytes at 37 °C occur below 5 M urea and 20% (V/V) ethanol, they should suffer only discrete spectral contributions of the effects of these chaotropes on oxyhemoglobin. CHAPTER III: Ethanol and urea are chaotropes that also exert actions as osmolytes. High concentrations of ethanol have been shown to promote the stabilization of erythrocytes. To better understand the origin of this stabilization effect, we have evaluated the influence of heat on hemolysis by urea and the influence of urea on hemolysis by ethanol and hypotonic stress. Sigmoidally-defined curves have been used to determine the half-transition points of hemolysis by urea (D50u), ethanol (D50e), and hypotonic stress (H50). Increasing temperature led to a decrease in the value of D50u, which may be ascribed to the synergistic chaotropic effects of heat and urea. Increasing urea concentration led to a decrease in the value of D50e, indicating a synergistic action of urea with ethanol in promoting hemolysis. Increasing urea concentrations led to decreasing values of H50, indicating that urea antagonized the hemolytic effect of hypotonic stress. The dependences of D50u on temperature and of D50e on urea concentration both produced sigmoid-shaped curves with broad transitions between two different regions of stability. The dependence of H50 on the concentration of urea was defined by a sigmoid-shaped curve with a sharp transition between two different regions of stability. This behavior can be attributed to an enhancement of erythrocyte stability induced by the combination of chaotropes.