Vibrational modes of hemoglobin in red blood cells.

Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective.     

Haloperidol reduction can be assayed in human red blood cells

One metabolite of haloperidol present in plasma is "reduced haloperidol." This study demonstrates that human red blood cells are capable of converting haloperidol to reduced haloperidol in vitro. The reductase involved requires NADPH, as does haloperidol (ketone) reductase in human liver cytosol.     

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.     

The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin.

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.     

活体红细胞内血红蛋白的电泳释放

当今的蛋白质组学研究,都是先裂解细胞放出蛋白质,然后对蛋白质溶液进行各种分析.对于红细胞来说,它的裂解产物也称＂溶血液＂,其中主要成分有血红蛋白A1,A2,A3和碳酸酐酶（CA）等.本实验室用未裂解的完整的活体红细胞直接进行电泳,观察其释放出来的血红蛋白（hemoglobin,Hb）,建立了淀粉-琼脂糖混合凝胶中红细胞的电泳释放实验.电泳释放可分为＂初释放＂（一次通电完成电泳,此时有Hb释放出来）和＂再释放＂（电泳过程中断电-再通电,又有Hb释放出来）.本实验室在＂初释放＂实验中发现了＂HbA2现象＂,并通过Hb交叉电泳发现了HbA2与HbA1的相互作用;利用初释放型双向对角线电泳发现红细胞内HbA2与HbA1结合存在;对电泳释放出来的＂HbA2现象＂成分做SDS-PAGE及质谱分析,发现Prx-2（Peroxiredoxin-2）可能参与＂HbA2现象＂的形成;在研究＂再释放＂实验中发现了＂Hb多带再释放现象＂,在此基础上创建等渗再释放、低渗再释放、等低渗全程再释放及再释放型双向对角线电泳;两种红细胞（全血中的红细胞和由它分离出来的游离红细胞）再释放的比较研究;血浆成分对红细胞再释放的影响等.以上研究方法的建立为活体细胞内蛋白质存在状态的研究提供了基础,并开辟了新的研究途径和领域.     

Prorenin is present in human red blood cells.

We looked for the presence of prorenin in erythrocytes from normal subjects (n = 8), hypertensive patients (n = 8), and pregnant women (n = 8). Angiotensin I generation was measured at 37 degrees C, pH 5.7, in the presence of homologous substrate (1400 ng/mL) before and after trypsin activation (100 micrograms/mL) in (A) haemolyzed erythrocytes, (B) supernatants of haemolyzed erythrocytes, and (C) in the sixth washing of erythrocytes diluted 1:1 with a 0.1 M Tris buffer containing 0.5% bovine serum albumin and protease inhibitors. Haemolyzed erythrocytes generated angiotensin I only after trypsin treatment, and the rate of generation was the same (A) before and (B) after centrifugation at 20,000g, indicating the absence of prorenin bound to the cell membranes. When aliquots of the last washing of erythrocytes (C) were tested for angiotensin I generation before and after trypsin, they did not generate angiotensin I, indicating that residual prorenin from the plasma was no longer present in our preparation. Angiotensin I generation by trypsin-treated A and B was completely abolished by preincubation with anti-renin serum. The level of prorenin was not significantly different in the erythrocytes from normal, hypertensive, and pregnant subjects (68 +/- 10, 58 +/- 7 and 107 +/- 17 pg angiotensin I.mL-1.h-1, ns) in spite of their very different plasma levels (21 +/- 2.5, 17 +/- 2.4 and 110 +/- 12 ng angiotensin I.mL-1.h-1, p less than 0.01 for pregnant women compared with both normal and hypertensive subjects). Our data show that prorenin is present in human erythrocytes in fairly constant and clearly detectable amounts, thus suggesting a possible intracellular role for it.     

Free radical-mediated platelet activation by hemoglobin released from red blood cells.

It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptor-based, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation of platelets.     

Cholesterol ester hydrolase in human red blood cells.

1. Cholesterol ester hydrolytic activity (sterol-ester hydrolase EC 3.1.1.13) was detected in human red blood cells. Enzyme activity appeared confined to the cell membrane and was most marked in washed preparations of red cell ghosts. 2. Hydrolytic activity was stimulated by the anti-oxidants D-alpha-tocopherol and butylated hydroxytoluene. Marked inhibition was produced by erythrocyte hemolysate, sodium taurocholate, and Triton X-100. 3. Optimal pH for the reaction was 5.4--5.7. 4. Because red cell cholesterol is all unesterified, it is speculated that the hydrolase serves to maintain the erythrocyte membrane free of esterified cholesterol.     

Microscopic diffusion and hydrodynamic interactions of hemoglobin in red blood cells

The cytoplasm of red blood cells is congested with the oxygen storage protein hemoglobin occupying a quarter of the cell volume. The high protein concentration leads to a reduced mobility; the self-diffusion coefficient of hemoglobin in blood cells is six times lower than in dilute solution. This effect is generally assigned to excluded volume effects in crowded media. However, the collective or gradient diffusion coefficient of hemoglobin is only weakly dependent on concentration, suggesting the compensation of osmotic and friction forces. This would exclude hydrodynamic interactions, which are of dynamic origin and do not contribute to the osmotic pressure. Hydrodynamic coupling between protein molecules is dominant at short time- and length scales before direct interactions are fully established. Employing neutron spin-echo-spectroscopy, we study hemoglobin diffusion on a nanosecond timescale and protein displacements on the scale of a few nanometers. A time- and wave-vector dependent diffusion coefficient is found, suggesting the crossover of self- and collective diffusion. Moreover, a wave-vector dependent friction function is derived, which is a characteristic feature of hydrodynamic interactions. The wave-vector and concentration dependence of the long-time self-diffusion coefficient of hemoglobin agree qualitatively with theoretical results on hydrodynamics in hard spheres suspensions. Quantitative agreement requires us to adjust the volume fraction by including part of the hydration shell: Proteins exhibit a larger surface/volume ratio compared to standard colloids of much larger size. It is concluded that hydrodynamic and not direct interactions dominate long-range molecular transport at high concentration.     

Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.     

Malonate transport in human red blood cells

Kinetic parameters of [2-¹⁴C]malonate uptake by the human erythrocyte membrane have been determined as K_m, 24 mM and turnover number, 5 × 10⁴ s^–1. The translocation of this organic dianion is concentration, pH and temperature dependent. Competitive inhibition of malonate uptake by eosin and inorganic anions, strongly implies that a common route exists for both inorganic anions and organic dianions, namely the anion-exchange Band 3 protein. ¹⁴C-Malonate which is nonmetabolized in the erythrocyte, could be a useful probe for monitoring anion-exchange in reconstituted Band 3 systems.     

Temperature transitions of protein properties in human red blood cells.

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.