Hemolysis of erythrocytes from human and porcine blood was studied at different calcium chloride (CaCl(2)) concentrations (0.04-1.83 mM) and at a constant bilirubin concentration (72 mM). Although, human erythrocytes showed significant hemolysis (64%) at the highest CaCl(2) concentration (1.83 mM) used in this study, remarkable resistance to this phenomenon was observed with porcine erythrocytes as only 11% hemolysis was observed. A similar pattern in the behavior of both human and porcine erythrocytes was observed when parameter such as bilirubin concentration or time of incubation with bilirubin was varied. Other divalent cations such as Mn(2+), Ba(2+) and Mg(2+) were either least effective or ineffective in inducing hemolysis in presence of bilirubin. Serum albumin played a protective role in this phenomenon in a concentration dependent manner, as no hemolysis was observed at a bilirubin/albumin molar ratio of 1:1 or less. Differences in the structural make-up of proteins and lipids in the erythrocyte membranes of the two species may account for their different behavior.
Interaction of bromophenol blue (BPB) with serum albumins from different mammalian species, namely, human (HSA), bovine (BSA), goat (GSA), sheep (SSA), rabbit (RbSA), porcine (PSA) and dog (DSA) was studied using absorption and absorption difference spectroscopy. BPB-albumin complexes showed significant differences in the spectral characteristics, i.e., extent of bathochromic shift and hypochromism relative to the spectral features of free BPB. Absorption difference spectra of these complexes also showed variations in the position of maxima and absorption difference (deltaAbs.) values. Absorption difference spectra of different bilirubin (BR)-albumin complexes showed a significant blue shift accompanied by decrease in deltaAbs. values in presence of BPB which were indicative of the displacement of bound BR from its binding site in BR-albumin complexes. These changes in the difference spectral characteristics of BR-albumin complexes were more marked at higher BPB concentration. However, the extent of these changes was different for different BR-albumin complexes. Taken together, all these results suggest that BPB partially shares BR binding site on albumin and different mammalian albumins show differences in the microenvironment of the BR/BPB binding site.