Rabbit Hemoglobin Research Articles

Initiation of rabbit hemoglobin synthesis: methionine and formylmethionine at the N-terminal.

Ribosome-bound peptides were prepared from rabbit reticulocytes incubated in a reaction mixture that contained all essential amino acids except tryptophan. The peptides were fractionated by Sephadex gel filtration and the N-termini of these peptides were examined. Longer uncompleted hemoglobin chains (larger than 30 amino acids) had unblocked valine at the N-terminal position. In contrast, shorter initial parts of chains (smaller than 16 amino acids) did not have valine at the N-terminal position. These small peptides had methionine at the N-terminal, and 8% of the terminal methionine was presumably formylated. These findings indicate that hemoglobin chains are initiated from methionine or N-formylmethionine (or from both methionine and N-formylmethionine), and that the methionyl residue is hydrolyzed at an early stage of chain elongation.

The amino acid sequence of the beta-chains of rabbit hemoglobin.

A partial formula of the primary structure of the β‐chain of rabbit hemoglobin is shown. Two variants of the β‐chain of rabbit hemoglobin have been found. One variant has β52His, β56Ser and β76Asn. The other variant has β52Asn, β56Asn and β76Ser. These variants are not strain specific.

Protein chain initiation in rabbit reticulocytes.

About 15% of the nascent alpha-chains isolated from in vivo labeled rabbit reticulocytes have methionine as their amino terminal amino acid. The methionine is predominantly on the shortest nascent chains, suggesting that methionine initiates the synthesis of rabbit hemoglobin and is rapidly removed during further synthesis.

An inheritable abnormal beta-chain in rabbit haemoglobin.

Mediante tecniche cromatografiche ed elettroforetiche, e state trovata una globina anormale nella emoglobina di un coniglio. La mutazione globinica riguarda verosimilmente laβ catena. Questa emoglobina anormale e trasmessa ereditariamente.

Cell-free transfer of leucine by transfer ribonucleic acid from mouse liver and plasma cell tumors into rabbit hemoglobin.

Cell-free transfer of leucine by transfer ribonucleic acid from mouse liver and plasma cell tumors into rabbit hemoglobin.

Methemoglobin formation by nitrobenzene in vitro

A small amount of methemoglobin was formed in vitro by incubating human or rabbit hemoglobin with nitrobenzene, a substance which has not been considered to form methemoglobin. After incubation with nitrobenzene, the oxidase activity of hemoglobin increased, and the alkali resistance of hemoglobin decreased. However, neither hemichromogen nor choleglobin was detected in the hemolysate after incubation. These facts show that nitrobenzene acts directly on and transforms the hemoglobin causing the heme iron to be changed to the ferric state. The molecular structure of methemoglobin was also discussed.

The distribution of specificity in rabbit antisera directed toward human hemoglobins

The distribution of specificity in antisera to human A 1 (α 2 Aβ 2 A and F (α 2 Aγ 2 F) hemoglobins was found to differ. In anti A 1 sera similar amounts of antibodies to α and β chains were found while in anti F sera there was always a preponderance ( 2 3 – 3 4 ) of antibodies to γ chains compared to α chains. In addition, hemoglobin F was found to be a more potent immunogen than hemoglobin A 1 and this superior immunogenicity was accounted for by the enhanced immunogenicity of the γ polypeptide chain. The sequence differences between the α, β, and γ chains of human hemoglobin and the α and β chains of rabbit hemoglobin were noted and correlated with the immunological findings.

Hemoglobine de lapin: une variante resultant d'un allelomorphisme et non d'une ambiguite: Hb α 29 Val → Leu

Our technic of preparative separation of two α-TIV, α-TIV Leu and α-TIV Val, from rabbit hemoglobin allowed us to described the following results. 1. 1. The relative amounts of α-TIV Leu and α-TIV Val vary non-continuously according to rabbits (0; 100; 50; 50; 100; 0). 2. 2. The biosynthesis in a cell-free system of reticulocyte containing one or two α TIV hemoglobin peptides occurs at the same relative rate. 3. 3. These findings, together with our previous conclusions about the information brought by mRNA from reticulocytes, with one α-TIV hemoglobin peptide type, to the ribosomes of reticulocytes, with another type, allow us to conclude that there are two allelomorphs rabbit hemoglobins: Hb α 29 Val and Hb α 29 Leu.

Allelic variants in the amino-acid sequence of the alpha chain of rabbit haemoglobin.

THE α chain of rabbit haemoglobin from a single rabbit does not always have a unique primary structure. Von Ehrenstein1 reported at least six positions in the α chain where alternative amino-acids can occur (Fig. 1); he suggested this variance was caused by translational ambiguity, rather than polymorphic alleles or multiple non-allelic genes. Analyses of similar variance in the a chain of goat2, horse (ref. 3 and personal communication by J. B. Clegg) and mouse haemoglobins4 and in the γ chain of human foetal haemoglobin5 indicate occurrence of non-allelic genes for these chains, but the variants in the β chain of adult human haemoglobin behave as true allelic genes6. We feel that the variants in the α chain of rabbits are linked to form two distinct linked sets which segregate in mendelian fashion, suggesting that the variance in rabbits is the result of polymorphic alleles of a single α chain gene.

Inhibition of the Biosynthetic Completion of Rabbit Hemoglobin by Isolated Human Hemoglobin Chains

Abstract A cell-free system from rabbit reticulocytes incorporated 14C-valine and 14C-leucine into hemoglobin. This synthesis of rabbit hemoglobin was inhibited in a specific manner when α or β chains, isolated from human hemoglobin, were added to the system. The human α and β chains interacted with the newly synthesized 14C-rabbit chains to form hybrid hemoglobins, which were identified by cellulose acetate electrophoresis, and the amount of 14C-rabbit hemoglobin was decreased. When human β chains were added to the reaction, there was also a 40 to 50% decrease in the amount of the 14C-rabbit β chain component in the supernatant fraction and an increase in the amount of completed 14C-β chains in the ribosome fraction, as determined by chain separation chromatography of these fractions on carboxymethyl cellulose columns. The supernatant phase also contained an electrophoretic fraction with the properties of free 14C-rabbit β chains; this fraction was not detected in the system incubated without human β chains. Addition of hemin at the beginning of the reaction largely prevented the change in distribution of β chain radioactivity between supernatant and ribosome fractions caused by human β chains. It is suggested that, during rabbit hemoglobin synthesis, a pool of α chains, endogenous to the soluble phase of the reaction, aided in the release or solubilization of some of the completed rabbit β chains from the ribosome fraction. Human β chains, when added to the reaction, interacted with the α chains to prevent them from aiding in this release. Under these conditions some of the newly synthesized rabbit β chains were released as free chains, while others remained bound to the ribosomes. Addition of hemin to the reaction prevented the accumulation of ribosome-bound chains by aiding in their release. The enhanced release of newly synthesized β chains by soluble α chains or hemin may be part of a translational control mechanism in hemoglobin assembly; however, the presence of free 14C-β chains in the supernatants of reactions containing human β chains suggests that α chains were not required absolutely for this release.

Specificity of yeast glutamic acid transfer RNA for codon recognition

Two species of tRNA for glutamic acid (glutamic acid tRNA's I and III) were separated from yeast tRNA, and the stimulation of their binding to Escherichia coli ribosomes by the trinucleotides GpApA and GpApG, which are known to be codons for glutamic acid, was tested. The specificity of these two species of glutamic acid tRNA's for codon recognition was further studied by testing the transfer of glutamic acids to the specific amino acid position in rabbit hemoglobin by these tRNA species. The results of these studies showed that yeast glutamic acid tRNA's I and III specifically recognized the glutamic acid codons GpApG and GpApA, respectively. The codons for glutamic acid on the hemoglobin mRNA were tentatively assigned. It is the first clear demonstration that a codon containing adenosine residue in the third letter was specifically recognized by a tRNA.

Concerning the kinetics of polypeptide synthesis on polyribosomes

AbstractThe kinetics of biosynthesis of polypeptides on polyribosomes is analyzed in accordance with a simple mathematical model. Each ribosome is assumed to block L adjacent. (m‐RNA) template sites but to move a distance of one, and only one, template site (nucleotide triplet) upon the addition of each monomer unit to the growing polypeptide chain bound to it. Solutions are sought, for the probability, nj(t), that a template possesses, at time t, a polypeptide chain that has reached degree of polymerization j.Several classes of steady‐state solutions are obtained via machine computation. These correspond to various choices of relative rates of initiation, polymerization along templates, and termination (release of completed chains from templates).Experimental data available from radioactive pulse labeling experiments are discussed. The data obtained by Dintzis, and by Winslow and Ingram, in studies of the synthesis of the α chain of rabbit hemoglobin and human hemoglobin, respectively, are consistent with steady‐state solutions obtained from the current theoretical calculations when the rates of initiation and termination are of comparable magnitude and rate‐determining. In this case, a (relatively) small number of chains are polymerized at a (relatively) fast rate each near the beginning of the template, and there exists a transition to a situation near the end of the template in which a (relatively) large number of chains are polymerized at a (relatively) slow rate each. For this solution the situation near the end of the template is entirely analogous to a traffic jam in automobile traffic.

Protective factors in the detoxication of hydrogen peroxide in erythrocytes.

The scope of catalase and of glutathione as factors protecting the hemoglobin of erythrocytes of several animal species against oxidation by hydrogen peroxide has been studied. In catalase-rich cells, catalase by itself can largely protect the hemoglobin even if glutathione is immobilized with iodoacetamide. The protective effect of added glucose was studied in erythrocytes exposed to hydrogen peroxide in the presence of the catalase inhibitors 3-amino-1,2,4-triazole and sodium azide. Except in the cow erythrocytes, glucose is uniformly effective in protecting hemoglobin and also in maintaining glutathione in the presence of aminotriazole. Lactate is generally ineffective in protecting hemoglobin or maintaining glutathione. Inhibition of catalase by azide makes the hemoglobin of monkey erythrocytes quite vulnerable to oxidation by hydrogen peroxide even in the presence of added glucose, but rabbit hemoglobin is effectively protected under the same conditions. However, glutathione levels are maintained in both. Iodoacetamide causes increased methemoglobin formation in glutathione-free hemolysates exposed to hydrogen peroxide in the presence of azide. The effect is largest in the rabbit and guinea pig hemolysates, while it is least in the monkey hemolysate. Cysteine and glutathione can reverse this effect. It would appear that besides catalase and glutathione, a nonglutathione sulfhydryl compound(s) might have a role in the protection of hemoglobin.

Evidence that the dissociation of human haemoglobin by p-mercuribenzoate is related to the structure of the β-chain

Human haemoglobin A, but not rabbit or C57B1 mouse haemoglobin, was separated by electrophoresis into α- and β-subunits after treatment with excess p-mercuribenzoate at pH 6. Sedimentation equilibrium analyses revealed that human haemoglobin treated in this way was dissociated partially into monomeric subunits at this pH; the rabbit and mouse haemoglobins did not dissociate to monomers. Experiments were performed to determine if the ability of haemoglobin A to dissociate into the individual chains could be correlated with the structure of either the α- or β-chain. The α 2 A β 2 RAB and α 2 RAB β 2 A hybrid haemoglobins were formed and subsequently isolated by electrophoresis on columns of polyacrylamide gel. Upon treatment with p-mercuribenzoate, the α 2 RAB β 2 A hybrid, like human haemoglobin A, dissociated into two electrophoretically separable components. The α 2 A β 2 RAB hybrid, like the rabbit haemoglobin, did not dissociate into components separable by electrophoresis. Similar results were obtained in experiments with hybrid haemoglobins containing human and C57B1 mouse chains. These data indicate that the ability of haemoglobin A to dissociate into α- and β-subunits is related to the structure of the β-chain, and are consistent with the hypothesis that the presence and position of the two cysteine residues in the human β-chain are important in the dissociation by p-mercuribenzoate. These results suggest that high yields of haem-containing α-chains from some animal haemoglobins may be obtained by forming a hybrid with human haemoglobin A and subsequently dissociating the isolated hybrid with p-mercuribenzoate.

Haemoglobins, XVII. The primary structure of the beta-chain of rabbit haemoglobin.

Haemoglobins, XVII. The primary structure of the beta-chain of rabbit haemoglobin.

Initiation of the alpha chain of rabbit hemoglobin.

The first step in the synthesis of all proteins in E. coli is the binding of N-formyl-methionine tRNA to a complex formed between a 30 S ribosome and a mRNA molecule (Nomura and Lowry, 1967). The N-formyl-methionine initiates the synthesis of the protein, becoming incorporated at the amino terminal end of the growing polypeptide chains (Capecchi, 1966). At some point in the synthesis of each protein either the formyl group or the formyl group and the methionine residue are cleaved off to expose the amino terminal residue of the finished protein. We made the assumption that the mechanism of chain initiation in mammalian cells would also result in the transient incorporation of an initiator molecule at the amino terminal position of the growing polypeptide chains. We decided to check on this assumption by studying the synthesis of the α chain of hemoglobin in rabbit reticulocytes. This system has two advantages:...

The ambiguities in the rabbit hemoglobin: Evidence for a messenger RNA translated specifically into hemoglobin °

Fractional residues of amino acids present in the αTIV peptide from the α chain of rabbit hemoglobin cannot be explained by an ambiguous translation of a unique template; they are the consequence of the translation of at least two different messengers RNA. Fractional residues of amino acids are present in several positions of the α chain of rabbit hemoglobin; the amino acid sequence of the rabbit α chain is not unique, as suggested by non integral values of amino acid residues and demonstrated by sequential degradation (Weisblum et al 1965, Von Ehrenstein 1966). The question arises whether the amino acid multiplicities are due to the ambiguous translation of a unique template, or to a multiplicity of α chain templates. In order to answer this question, we have investigated the role of amino acyl tRNA, activating enzymes, and mRNA, in the ambiguity of the peptide αTIV of rabbit hemoglobin. The experimental evidence demonstrates the role of the template.

Evidence for Dimorphism in Rabbit Hemoglobins

SummaryThe hemoglobin of 24 rabbits was analyzed by the “finger-printing” technique and amino acid analyses. The data revealed the presence of dimorphism in rabbit hemoglobin. The difference in composition and/or structure appears to be located in the β-chain.

Polyribosomal changes during inhibition of rabbit hemoglobin synthesis by an isoleucine antagonist.

Polyribosomal changes during inhibition of rabbit hemoglobin synthesis by an isoleucine antagonist.

Biosynthesis of the Peptidoglycan of Bacterial Cell Walls: X. Further Study of the Glycyl Transfer Ribonucleic Acids Active in Peptidoglycan Synthesis in Staphylococcus Aureus

Abstract Three glycine acceptor transfer RNAs (tRNAs) have been separated from the crude tRNA of Staphylococcus aureus H. All three support the incorporation of glycine into peptidoglycan. Two of them participate in polypeptide synthesis as examined by various methods. However, one of the glycyl-tRNAs is inactive in polynucleotide-directed polypeptide synthesis as well as in the synthesis of rabbit hemoglobin. This tRNA does not bind to ribosomes in the presence of any of the known codons for glycine.