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D Guillochon

Publications and source records attributed to D Guillochon.

36 records · Page 2Linked to original sources
PubMed1995

Isolation from bovine haemoglobin of a peptide that might be used as a potential hydrophobic photosensitizer carrier.

The isolation of a peptic hydrolysate and of a pure peptide from bovine haemoglobin is described. These peptides were used to solubilize an insoluble photosensitizer in order to study their utilization as a carrier for photochemotherapy. Protoporphyrin IX was used as a model of an insoluble photosensitizer. Solubilization of protoporphyrin IX was first performed with the total haemoglobin hydrolysate, then with peptide fractions, and finally with a pure peptide isolated from this fraction by reversed-phase HPLC. The molecular mass and the primary structure of the pure peptide were determined by fast-atom-bombardment and tandem MS (molecular mass 1648 Da). The singlet oxygen quantum yield of the protoporphyrin IX-peptide complex was determined.

Amino Acid Sequence↗
PubMed1994

Photophysical and photobiological activities of a porphyrin peptide fraction derived from haemoglobin.

In a previous study, we described the preparation of a porphyrin peptide hydrolysate from haemoglobin, its isolation and its analysis by high performance liquid chromatography (HPLC) and fast atom bombardment (FAB) mass spectrometry. The purpose of the present paper is to test the photosensitizing activity of this fraction. We determined the singlet oxygen quantum yield (phi delta) in order to quantify the efficiency of the porphyrin peptide fraction. The quantum yield is about phi(1O2)=0.06. An analysis of the phototoxic effect on tumour cells in culture was performed and compared with haematoporphyrin derivative (HpD), the only photosensitizer in clinical use at present. The phototoxicity of the porphyrin peptide fraction is weaker than that of HpD. However, for a porphyrin dose of 50 micrograms ml-1, the difference in phototoxicity is low, and in the absence of irradiation porphyrin peptides are less toxic than HpD. These results suggest that porphyrin peptides could be potent photosensitizers; moreover, they are of great interest since they allow the solubilization of hydrophobic porphyrins and could be applied in the future as insoluble photosensitizer carriers.

Cell Line↗
PubMed1993

Stabilizing effect of water/alcohol solvents towards autoxidation of human haemoglobin.

The stabilization of haemoglobin (Hb) in water/alcohol solvents (ethanol, butan-1-ol, ethylene glycol and glycerol) against autoxidation, a major barrier to the successful use of Hb in biological and medical engineering was studied, with these solvents, a large decrease in the autoxidation rate is observed over a range of concentrations. Studies on the effect of Hb concentration on autoxidation showed that, irrespective of the Hb concentration, oxyHb is more stabilized in water/alcohol solvents than in water. In these solvents, at the concentration exhibiting the maximal stabilizing effect, the oxygen affinity of Hb was little disturbed, but the changes in enthalpy and entropy of activation increased with the stabilization effect of alcohol. The impact of alcohols on the thermal denaturation of metHb was studied. We observed that metHb is less stable in ethanol and butan-1-ol than in water, whereas the opposite is observed with glycerol and ethylene glycol. The strong stability of oxyHb observed with these solvents could result both from an increase in water structure and an increase of viscosity. Finally, e.p.r. has shown that alcohols lead to an increase of the global distance between the nitrogens of proximal histidine (F8) and nitric oxide in the nitrosyl Hb.

1-Butanol↗
PubMed1992

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a bradykinin-potentiating peptide from a bovine peptic hemoglobin hydrolysate.

A bradykinin potentiating peptide was isolated from a peptic bovine hemoglobin hydrolysate, by the use of reversed-phase high-performance liquid chromatography (RP-HPLC). Its primary structure, determined by fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), was identical to fragment 129-134 of the alpha-chain of bovine hemoglobin. The bradykinin potency of this peptide, as exhibited by the guinea-pig ileum contraction, was significant and comparable with some others previously described.

Amino Acid Sequence↗
PubMed1991

Stabilizing effect of organic solvents on oxyhemoglobin.

The role of hemoglobin solutions as oxygen carriers in biotechnology are numerous, such as in the oxygen supply to biocatalysts or in the preparation of blood substitutes. However, the major barrier to the successful use of hemoglobin in biological and medical engineering is the autoxidation of heme iron during preparation, storage, and utilization. Fifty-six solvents, chosen among the group of Parker's classification, were studied with regard to the autoxidation kinetics of oxyhemoglobin under nondenaturant conditions. Among these solvents 27 present a concentration range in which the autoxidation rates were reduced compared to autoxidation in water. Three groups of solvent have been observed: one exhibiting only a destabilizing effect regardless of the solvent proportion, a second showing a strong stabilizing effect (k(H2O)/k(solvent) greater than 20) and a third showing a low stabilization (k(H2O)/k(solvent) less than 20). The most effective stabilizing solvents were glycerol, glycols, and alcohols. The effect of hydroorganic solvents could be explained by taking into account the globin solvation by water molecules. The solvents that enhance the structure of the water and form few hydrophobic interactions with globin prevent oxyhemoglobin autoxidation.

Humans↗
PubMed1990

Glutaraldehyde effect on hemoglobin: evidence for an ion environment modification based on electron paramagnetic resonance and Mossbauer spectroscopies.

Glutaraldehyde is a widely used reagent for hemoglobin cross-linking in blood substitutes research. However, hemoglobin polymerization by glutaraldehyde involves modifications of its functional properties, such as oxygen affinity, redox potentials, and autoxidation kinetics. The aim of this article is to investigate, by electron paramagnetic resonance and Mossbauer spectroscopies, the changes that occur in the iron environment after glutaraldehyde cross-linking. Spectrometric studies were performed with native hemoglobin and hemoglobin cross-linked as soluble and insoluble polymers. Spectrometry data comparison with glutaraldehyde-modified hemoglobin functional properties allows to interpret from a structural point of view that glutaraldehyde action occurs as a decrease of the O--N(F8His) distance, an increase of the Fe--N(F8His) bond length, and the decrease of the distal-side steric hindrance.

Cross-Linking Reagents↗
PubMed1989

Identification of peptides, from a peptic haemoglobin hydrolysate produced at pilot-plant scale, by high-performance liquid chromatography and mass spectrometry.

Gel-permeation high-performance liquid chromatography (HPLC) and reversed-phase HPLC were used to separate a mixture of peptides, produced at pilot-plant scale by peptic hydrolysis of bovine haemoglobin. Volatile buffers were employed in both HPLC techniques in order to get an easy recovery of peptides for further applications. The method is more rapid than low-pressure gel filtration. Amino acid analysis and fast atom bombardment mass spectrometry confirmed the purity, and allowed accurate molecular weights to be determined, for isolated peptides. These data demonstrate that such efficient techniques, usually used to resolve hydrolysates obtained in batch with pure substrates and highly specific enzymes, can be employed to resolve complex enzymatic hydrolysates of crude protein.

Amino Acid Sequence↗
PubMed1986

Effect of glutaraldehyde on haemoglobin: oxidation-reduction potentials and stability.

Glutaraldehyde is a reagent widely used for the cross-linking of haemoglobin for use as a blood substitute. Most of the previous studies were limited to oxygen binding equilibria of the glutaraldehyde-modified haemoglobin. This paper concerns the impact of glutaraldehyde on oxidation-reduction equilibria, autoxidation kinetics and stability towards heat and urea of haemoglobin cross-linked in the oxy, deoxy and ferri states. The oxidation-reduction potentials and homotropic effects were reduced; however, the oxidation Bohr effect was not significantly different when compared with native haemoglobin. Haemoglobin immobilized in the oxy or ferri state exhibited a lower redox potential than when immobilized in the deoxy state. The autoxidation rates were increased after cross-linking, particularly at basic pH. Cross-linking stabilizes ferrihaemoglobin better than oxy or deoxyhaemoglobin against thermal- and urea-induced denaturation. Glutaraldehyde cross-linking does not stabilize haemoglobin against urea-denaturation. The experimental results were interpreted as indicating a chemical modification of the protein without 'conformation freezing' and by an opening of the haem pocket to the aqueous solvent.

Aldehydes↗
PubMed1986

Effect of glutaraldehyde on hemoglobin: functional aspects and Mössbauer parameters.

Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with Mössbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation-reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by Mössbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.

Aldehydes↗
PubMed1986

Aromatic hydroxylations in peroxidations by haemoglobin systems.

The catalytic activity of haemoglobin on aromatic substrates was studied in three systems: NADH-methylene blue-haemoglobin, ascorbic acid-haemoglobin, and red blood cells. Aniline and phenol but not acetanilide or p-toluidine are hydroxylated by haemoglobin. Dealkylations are not observed. Hydroxylations are postulated to be intermediate reactions in peroxidations catalysed by haemoglobin. The lifetime of the products depends on the presence of electron donors, such as NADH or ascorbic acid, in the medium. In the red blood cells where endogenous electron donors are recycled, levels of the products are higher and their lifetime is longer. This could have implications on drug metabolism by haemoglobin, as haemoglobin is present in large quantities in the organism.

Animals↗
PubMed1985

Determination of metabolites of cytochrome P-450 model systems using high-performance liquid chromatography.

High-performance liquid chromatographic techniques were developed for the simultaneous detection of metabolites in a cytochrome P-450 model system composed of NADH, haemoglobin and methylene blue. Monohydroxylated metabolites were determined following aniline, acetanilide and phenol hydroxylations. 4-Aminoantipyrine, 7-hydroxycoumarin and p-nitrophenol were determined after dealkylation of 4-N,N-dimethylamino-antipyrine, 7-ethoxycoumarin and p-nitroanisole. These substrates are commonly used for measuring cytochrome P-450 activities. Treatment of the samples was minimal, consisting of a simple deproteinization, and did not involve any organic extraction. Separations were carried out on reversed-phase columns and the products were detected by UV adsorption. Separations were completed in less than 15 min and the detection limits were between 0.5 and 4 microM.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1981

Studies on haemoglobin immobilized by cross-linking with glutaraldehyde. Cross-linked soluble polymers and artificial membranes.

Human haemoglobin was immobilized by cross-linking with glutaraldehyde as soluble polymers and artificial membranes. Effects of pH and 2,3-diphosphoglycerate on oxygen binding and cross-linking were studied with haemoglobin immobilized in both the oxy and deoxy states. The cooperativity is suppressed and the affinity is increased when compared with native haemoglobin. Haemoglobin immobilized in the oxy state exhibited a higher oxygen affinity than that immobilized in the deoxy state. The alkaline Bohr effect is not significantly different from that of native haemoglobin. The 2,3-diphosphoglycerate influence on oxygen binding was reduced by one third with immobilization. In order to separate the chemical and the "conformation freezing' effects on the properties of immobilized haemoglobin, glutaraldehyde-modified haemoglobin in oxy and deoxy states was produced. Oxygen binding was studied and chemical modifications were checked by electrophoresis and gel filtration. This chemically modified haemoglobin without polymerization and without intra-chain bridging exhibits a behaviour similar to that of cross-linked soluble polymers or membranes of haemoglobin.

2,3-Diphosphoglycerate↗
PubMedDate not supplied

Effect of stabilising amino acids on the digestive absorption of heme and non-heme iron.

We used the Ussing chamber model to study heme iron absorption by rat duodenal mucosa. Heme iron was obtained by enzymic digestion of bovine haemoglobin and concentration of heme (HPH). Its uptake and mucosal transfer was compared to iron gluconate (Gluc), at 100 microM and 1 mM. At 100 microM iron uptake (Qtot), mucosal retention (Qm) and transfer across the mucosa (Qs) was similar for the two sources of iron. Qs was significantly higher at 1 mM for Gluc but not for HPH, and was associated with higher levels of Qm. Addition of L-histidine did not improve iron absorption and indeed it decreased it if iron was provided as Gluc. L-cysteine increased the transfer of iron of both sources. In the in vitro model using rat digestive mucosa, heme iron appeared to be an efficiently used source of iron, which might prevent its accumulation by gut when supplied in excess.

Amino Acids↗