A.G. Schnek

Oxygen affinity of avian hemoglobins

Abstract 1. 1. Blood from different avian species displays the presence of either two major hemoglobin components, type 1 and type 2, or a unique one similar to type 2. 2. 2. Some aspects of oxygen equilibrium are reported for both types of hemoglobin obtained from several species, namely oxygen affinity in the absence or presence of inositolhexaphosphate, the Bohr effect and the heme-heme interaction. 3. 3. The analogies and differences illustrated at the level of the physicochemical parameters are also observed at the level of the physiological properties.

Purification and some molecular properties of protein methylase II from equine erythrocytes

Abstract Protein methylase II (S-adenosyl-L-methionine: protein carboxyl-O-methyltransferase; E.C. 2.1.1.24) has been purified 28 000 fold from equine erythrocytes. The purified enzyme is homogeneous on polyacrylamide gel electrophoresis performed either in presence or in absence of SDS, and on analytical ultracentrifugation. It appears constituted of a single polypeptidic chain of a molecular weight very close to 25 000 Daltons. Other enzymatic properties of the protein are quite similar to those previously reported for similar enzymes. The amino acid analysis of the enzyme is presented. The single cysteine residue, the enzyme contains, is essential for the enzymatic activity. Other amino acids apparentely involved in catalysis are tentatively identified.

The primary sequence of chicken myoglobin (Gallus gallus)

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.

Evidence that trimethylation of iso-I-cytochrome c from Saccharomyces cerevisiae affects interaction with the mitochondrion

Cytochrome c is a protein occurring in all eucaryotic organisms. Its function and gross conformation has remained the same through evolution as indicated by the constancy of properties like its absorption spectrum, biological activity and redox potential [l]. Of the 103 amino acid ‘core sequence’ common to all types of cytochromes c so far sequenced 35 residues are found to be constant [2]. The longest invariant sequence is the undecapeptide comprising residues 70-80. This segment is thought to be very important for the function of the haemoprotein: indeed the sulfur atom of methionine 80 is most probably the sixth ligand to the haeme iron, tyrosine 74 participates to the mechanism of reduction of the haemoprotein by succinate cytochrome c oxidoreductase [3] and lysines 72 and 79 appear to be involved in binding with cytochrome c oxidase [4]. Yet, the undecapeptide 70-80 is not strictly invariant in all cytochromes c, since cytochrome c from Ascomycetes and higher plants has the lysine 72 e-N-trimethylated [5 1. The biological role of this posttranslational modification is still unclear and has been proposed [6] that methylation of lysine 72 is more coincidental than specific. In investigations comparing the methylated and

Penguin (Aptenodytes forsteri) myoglobin. A 70 residue N-terminal sequence

Since Edmundson [1] determined the amino acid sequence of the sperm whale myoglobin in 1965, this protein has been examined in several other species ( land and marine mammals, gastropod). With the purpose of elucidating evolutionary and genetic informations, comparative structural studies have been extended to a new zoological class: the birds. The partial covalent structure of chicken myoglobin was determined [2] first. This paper presents the 70 residue N-terminal sequence of the penguin myoglobin. Residues have been positioned after sequence data obtained on peptides isolated from tryptic (T), chymotryptic (C), tryptic following maleylation of the protein (TM) and cyanogen bromide (CNBr) digestions of the globin. These results have been confirmed by stepwise degradation of the whole globin and of a large TM peptide using a sequencer.

N terminal amino acid sequences of the α and β chains of the two chicken hemoglobin components

Abstract Amino acid sequences corresponding approximately to the N-terminal third of the α and β chains of the minor chicken hemoglobin component (P1) have been determined. When each subunit is compared to the corresponding chain belonging to the major chicken hemoglobinic component (P2), a high number of amino acid substitutions can be observed between both chicken α chains while no difference can be noticed between the two β subunits.

Isolation and characterization of chicken (Gallus gallus) and penguin (Aptenodytes forsteri) myoglobins

Summary Methods describing isolation, purification and characterization of two avian myoglobins are reported. Chicken myoglobin was extracted at 55 to 90 p. cent ammonium sulfate saturation, while penguin myoglobin was isolated by low temperature fractionation using ethanol in the presence of metallic ions. Both were purified by gel filtration and chromatography on CM Sephadex and their homogeneity was tested by zone electrophoresis with different gels. The amino acid compositions have been determined. Chicken hemoprotein appears very similar to previously studied myoglobin, but penguin hemoprotein differs significantly, essentially by a higher amount of methionine. Both proteins have glycine as the aminoterminal residue. The molar extinction coefficients and the reduced mean residue optical rotation were found to be identical.

Penguin hemoglobin (Aptenodytes forsteri). A 45 residue N terminal sequence

The hemoglobins have been studied from the point of view of their amino acid sequence principally in mammals; this study has also been extended to the fish (carp, lamprey) and to the invertebrates (chironomus thummi, Glycera dibranchiata). Nevertheless, as far as birds are concerned, only the primary structure of the o-chain of the chicken major hemoglobin component is known at the present time. With the goal of expanding the bulk of knowledge with respect to structural comparative studies, the determination of the amino acid sequence of the o-chain of the penguin hemoglobin was undertaken. The positions of the first 45 residues of this (Ychain were determined by two different approaches: on the one hand from information obtained on the tryptic peptides of the two cyanogen bromide fragments of the a-chain, and on the other hand from results of the degradation of the entire polypeptide chain by use of the sequencer.

Sulfhydryl groups of chicken hemoglobins. Effect of the reaction with para-mercuribenzoate on subunits dissociation and oxygen affinity

Summary After quantitative reaction of the sulfhydryl groups of chicken hemoglobins with para mercuribenzoate, α and β chains cannot be separated by chromatography from either major or minor component. Ultracentrifugations show that dissociation into dimers occurs, but at protein concentrations much lower than those observed for mammalian hemoglobins. Oxygen equilibrium is affected: cooperative effects are weakened and oxygen affinity increases, the amplitude of the alteration being different for each component in presence or in absence of inositol hexaphosphate. Comparison with the behaviour of some mammalian hemoglobins is discussed.

Reoxidation of reduced hen egg white lysozyme fragment 1-123.

The reactivation of reduced lysozyme, whose 6 COOH-terminal amino acid including cysteine 127 were cut off, was studied. The results show that the disulfide bridge I–VIII as well as the COOH-terminal hexapeptide do not play a decisive role in the acquisition of the native 3-dimensional structure of the enzyme.