Ultrasonic absorption and relaxation spectra in aqueous bovine hemoglobin

Measurements of the frequency and p^H dependence of acoustic absorption at 0°C in aqueous solutions of freshly prepared bovine oxyhemoglobin are reported. The role of ionization and possible direct proton-transfer between proximal pairs in determining the characteristic times for the relaxation of the internal charge distribution is discussed. It is concluded that treatments which consider various classes of residues as ionizing independently will not give approximately correct relaxation spectra. A model which takes into account the coupling between the degrees of ionization of the various residues is found to give rough agreement with the observed acoustic absorption in the p^H range in which the native conformation is stable.     

Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy.

When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed.     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

The indirect effect of hemoglobin on the electrophoretic mobility of human blood erythrocytes

Using the factor analysis, it was shown that the total content of hemoglobin in human blood plays a limited and indirect role in the regulation of average electrophoretic mobility of erythrocytes. In this case, it is not the only parameter governing this level. The statistical relationship between the total content of hemoglobin and erythrocyte mobility in electrical field is not stable and is determined by the dependence of both indices on their common factor of control of erythroid homeostasis.