Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history

Primitive erythroblasts in the circulating blood of the chick embryo continue to divide while synthesizing hemoglobin (Hb). Hb measurements on successive generations of erythroblasts show that there is a progressive increase in the Hb content of both interphase and metaphase cells. Furthermore, for any given embryo the Hb content of metaphase cells is always significantly greater than that of interphase cells. The distribution of Hb values for metaphase cells suggests that there are six Hb classes corresponding to the number of cell cycles in the proliferative phase. The location of erythroblasts in the cell cycle was determined by combining Feulgen cytophotometry with thymidine radioautography on the same cells. Measurements of the Hb content for erythroblasts in different compartments of the cell cycle (G1, S, G2, and M) show a progressive increase through the cycle. Thus, the amount of Hb per cell is a function of the number of cell divisions since the initiation of Hb synthesis and, to a lesser degree, the stage of the cell cycle. Earlier generations of erythroblasts synthesize Hb at a faster rate than the terminal generation. Several models have been proposed to explain these findings.     

Simulating transmeridian flights

Sleep and Biological Rhythms -     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.     

Mouse hemoglobin during gestation. Absence of fetal hemoglobin

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.     

The inhibition of hemoglobin C crystallization by hemoglobin F

We have reported that circulating CC erythrocytes containing HbO2 C crystals exhibit little or no Hb F suggesting that Hb F may inhibit the crystallization of Hb C. We report now that Hb F inhibits in vitro crystallization of HbO2 and HbCO C when compared to the effect of Hb A in a wide range of mixture proportions. For example, while HbCO C solutions form tetragonal C crystals within 25 min, no crystals form within 2 h with 30% Hb F, whereas 550 crystals/mm3 form with 30% Hb A. Furthermore, an increase in the percent of Hb A is correlated with a greater number of orthorhombic crystal formation rather than the tetragonal morphology observed with 100% Hb C. We also report that Hb A2 (containing delta chains that exhibit 10 sequence differences with beta chains) and Hb Lepore Boston-Washington (a fusion mutant of delta and beta chains that contains only six of these differences) both inhibit Hb C crystallization. By comparing the sequences of the three inhibitory hemoglobins, we conclude that position Gln-87 in the gamma chains is, at least partially, the cause of the inhibitory effect of Hb F on the crystallization of Hb C.     

Alternative diaspirins for modification of hemoglobin and sickle hemoglobin

Studies of modification of hemoglobin and of sickle hemoglobin by alternative aspirins have been extended to a series of new bis esters with a variety of substituted bridging diacids and to a group of mono esters with polar acyl groups. Rates of hydrolysis of these alternative aspirins have also been examined, and they reveal that a careful balance between stability and reactivity is essential for optimal activity. Four-carbon bridging groups have been found to be particularly effective, two of these raising the minimum gelling concentration of sickle hemoglobin by as much as 100%.     

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)