José Leonis

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.

A microcalorimetric method of determination of critical micellar concentration and enthalpy of micellization

ZusammenfassungEine Methode zur Analyse der Verdünnungs-enthalpien von mizellaren Systemen wird vorgeschlagen. Messungen bei einer einzigen Temperatur ermöglichen gleichzeitig die Bestimmung der kritischen Mizellkonzentration und der Enthalpie der Mizellenbildung.Angewandt auf ionische und nichtionische oberflächenaktive Substanzen, ergibt diese Methode Resultate, die in guter Übereinstimmung sind mit den durch andere Autoren angegebenen Werten für wässerige Lösungen dieser Substanzen bei 25 °C.Die Bestimmung dieser beiden Parameter wird also in jedem beliebigen als Lösungsmittel benutzten Medium ermöglicht; wir haben unsere Messungen auch auf den Fall von oberflächenaktiven Substanzen in NaCl-Lösungen ausgedehnt.SummaryA method is proposed for the analysis of dilution heats of micellar systems. Simultaneous determination of critical micellar concentration and of micellization enthalpy can be derived from measurements performed at a single temperature.This method, when applied to ionic and non-ionic detergents, supplies results in good agreement with those published for detergents in aqueous solutions at 25 °C.Determination of these two parameters should thus be possible for any type of solvent; further measurements were extended to detergents in solutions 0.001, 0.01 and 0.1 M in NaCl.

Comparative physicochemical studies of human α-lactalbumin and human lysozyme

Abstract As a result of the recent disclosure of the striking similarities between the covalent structures of bovine α-lactalbumin and of hen egg-white lysozyme, comparative physicochemical properties of α-lactalbumins and lysozymes of various sources seemed worth investigating. Therefore human milk α-lactalbumin and human milk or urinary lysozyme were purified. Their physical properties were examined in order to study the extent of similarity of the three-dimensional structure when considering the two proteins from the same species. Diffusion and sedimentation experiments showed that the hydrodynamic shape and the molecular weight of human α-lactalbumin and lysozyme were strikingly comparable. Concerning the secondary structure, it was observed by optical rotatory dispersion and circular dichroism that human lysozyme displays slightly more helix than human α-lactalbumin. Furthermore, when considering thermal denaturation, the transition temperatures and changes in enthalpy or entropy are all markedly lower for human α-lactalbumin than for lysozyme. These findings have been discussed in relation to the physicochemical properties of bovine α-lactalbumin and egg-white lysozyme.

Cytochrome C methylation

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.

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.

Study of the biological significance of cytochrome methylation I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c

The iso-cytochromes c from bakers yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from bakers yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from bakers yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetess cytochromes as compared to mammalian ones.

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

Biological significance of methylation of cytochromes from ascomycetes and plants

Abstract The K m and V max values characterizing the reaction of bakers yeast iso -I-cytochrome c , whether tri-methylated or not at lysine residue 72, with crude preparations of cytochrome c peroxidase, cytochrome c oxidase and succinate cytochrome c oxidoreductase from Saccharomyces cerevisiae are similar. These results, as well as the redox potential values, the auto-oxidability parameters and the circular dichroism spectra, strongly suggest that the biological methylation of yeast cytochrome c does not alter its functional properties. The functional characteristics of bakers yeast iso -I-cytochrome c are similar to those of horse heart cytochrome c and yeast iso -2-cytochrome c.

Surface tension measurements by the drop-weight method for continuously varying surfactant concentration

A method is described to measure rapidly the superficial tension of any liquid, and to measure it with precision as a function of a continuous variation f the solute concentration. The method has been tested with 3 different surfactants. The first is an anionic one, sodium dodecyl sulfate (SDS); the second is a cationic one, cetyltrimethylammonium bromide (CTAB); and the last is a nonionic one, 1-(1,1-dimethyl-3,3-dimethylbu polyethylenoxybenzene (TX-100).