Kinetics of sickle hemoglobin polymerization. I. Studies using temperature-jump and laser photolysis techniques

Using a combination of laser photolysis and temperature-jump techniques, the kinetics of hemoglobin S polymerization have been studied over a wide range of delay times (10(-3) to 10(5)s), concentrations (0.2 to 0.4 g/cm3) and temperatures (5 to 50 degrees C). A slow temperature-jump technique was used to induce polymerization in samples with delay times between 10(2) seconds and 10(5) seconds by heating a solution of completely deoxygenated hemoglobin S. For samples with shorter delay times, polymerization was induced by photodissociating the carbon monoxide complex in small volumes (10(-9) cm3) using a microspectrophotometer equipped with a cw argon ion laser. The photolysis technique is described in some detail because of its importance in studying hemoglobin S polymerization at physiological concentrations and temperatures. In order, to establish conditions for complete photodissociation with minimal laser heating, a series of control experiments on normal human hemoglobin was performed and theoretically modeled. The concentration dependence of the tenth time is found to decrease with increasing hemoglobin S concentration. In the range 0.2 to 0.3 g/cm3, the tenth time varies as the 36th power of the hemoglobin S concentration, while in the range 0.3 to 0.4 g/cm3 it decreases to 16th power. As the tenth times become shorter, the progress curves broaden, with the onset of polymerization becoming less abrupt. For tenth times greater than about 30 seconds, measurements with the laser photolysis technique on small volumes yield highly irreproducible tenth times, but superimposable progress curves, indicating stochastic behavior. The initial part of the progress curves from both temperature-jump and laser photolysis experiments is well fit with an equation for the concentration of polymerized monomer, delta (t) = A[cosh (Bt) -1], which results from integration of the linearized rate equations for the double nucleation mechanism described in the accompanying paper (Ferrone et al., 1985). The dependence of the parameters A and B on temperature and concentration is obtained from fitting over 300 progress curves. The rate B has a large concentration dependence, varying at 25 degrees C from about 10(-4) S-1 at 0.2 g/cm3 to about 100 s-1 at 0.4 g/cm3.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.     

Characterization of hemoglobin Burke [β107 (G9) Gly→Arg]

Hb Burke [107 (G9) GlyArg] was discovered in a young woman with hemolytic anemia. A substitution in this position has not been previously reported either in the human -chain or in any of the animal -chains so far sequenced. The abnormal hemoglobin shows heat instability and a lowered oxygen affinity. The substitution of a large charged arginine residue for the small glycine residue in the G helix next to a heme contact (Leu-106) may be responsible for these effects. Hb Burke is compared with five other hemoglobins having Gly-Arg substitutions in other parts of the molecule.This work was supported in part by U.S. Public Health Service Grants AM-17850 and AM-18006.     

Hb Mobile [α2β2 73(E17)Asp → Val]: A new variant

A new hemoglobin variant found in a mother and her child was characterized by column chromatography of the tryptic hydrolysate of the aminoethylated, glycinamidated -chain, followed by chymotryptic digestion of the abnormal T-9 peptide and amino acid analyses. It was shown to be _{2 2} 73(E17) Asp Val and named Hb Mobile.This work was supported in part by Research Grants AM0780 and AM13173 from the National Institute for Arthritis and Metabolic Disease.     

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.     

Unveiling the Timescale of the R-T Transition in Human Hemoglobin

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place ∼ 2 μs after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-μs step observed with time-resolved optical spectroscopy corresponds to a small and localized structural change.     

Ligand binding and the gelation of sickle cell hemoglobin.

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm^?3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure.The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t_d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t_d for each 10% increase in the fractional saturation.These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.     

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (R_f) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (R_f × 100) was found to be directly proportional to the pK′₂ of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl-alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.     

Hemodynamic response of the frontal cortex elicited by intravenous thiamine propyldisulphide administration

The intravenous olfaction (IVO) test is a unique type of clinical olfactometry and is widely used in Japan. However, it is difficult to distinguish actual olfactory disturbance from feigned disturbance because the IVO test is a psychophysical test. To resolve this problem, we investigated the possibility of an objective IVO test assisted with near infrared spectroscopy (NIRS). IVO testing was performed according to the usual protocol with thiamine propyldisulphide (alinamin) administration. The relative oxy- and deoxyhemoglobin levels of the orbitofrontal area during olfactory stimulation by IVO test were measured by NIRS. Pairs of NIRS emitters and detectors were positioned on the bilateral frontal scalp. After administration of alinamin, oxyhemoglobin levels increased, though deoxyhemoglobin levels did not change. An increase in oxyhemoglobin levels was observed bilaterally. Administration of saline did not elicit any change in the oxy- or deoxyhemoglobin levels and concentration of the administered alinamin related increasing of the oxyhemoglobin level was observed. Oxyhemoglobin remained unchanged in anosmic subjects despite administration of alinamin. The latency of oxyhemoglobin increase on each side and smelling latency showed significant correlation. Latencies of oxyhemoglobin increases between the right and left sides also showed significant correlation. Oxyhemoglobin response appears to be linked to olfactory related response. NIRS is a useful technique for the development of an objective form of IVO testing.     

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl⁻) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl⁻ was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

Multiple immunoreactive forms of calcitonin in human plasma

Purified rabbit serum haptoglobin was partially characterized, and it was found that the hemoglobin-binding property and subunit structure were similar to those of human type 1-1, swine, canine, and equine haptoglobins. However, rabbit haptoglobin was dissociated into subunits and few intermediates only in the presence of urea or sodium dodecyl sulfate without reduction. Thus the absence of interchain disulfide bonds in rabbit haptoglobin is unique among many animal haptoglobins.