Didier Guillochon

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.

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 α‐chain of bovine hemoglobin. The bradykinin potency of this peptide, as exhibited by the guinea‐pis ileum contraction, was significant and comparable with some others previously described.

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.

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.

Hydroxylase activity of immobilized haemoglobin

Abstract Until now, hydroxylation of substrates for practical applications has been mostly carried out by chemical and microbial processes. The hydroxylase activity of haemoglobin could be of great help for this purpose. Hydroxylation of aniline by haemoglobin immobilized as cross-linked soluble polymers and insoluble particles was studied. Activity yields after immobilization as well as kinetic constants were estimated. Hydroxylase activities similar to those of liver microsomal cytochrome P -450 activities were obtained.

Aniline hydroxylase activities of haemoglobin: Kinetics and mechanism

Abstract The mechanism of the aniline hydroxylase activity of methaemoglobin in a monooxygenase system consisting of NADH as electron donor, riboflavin, FAD, FMN or methylene blue as electron carrier and methaemoglobin as the terminal oxidase has been studied. Hydrogen peroxide is produced from oxygen in a methaemoglobin-independent process. 4-Aminophenol is subsequently produced peroxidatively by an NADH-dependent process; NADH prevents a further oxidation of 4-aminophenol in the presence of haemoglobin. In the absence of electron carrier, NADH slowly reduces haemoglobin and then oxyhaemoglobin reacts with aniline to give 4-aminophenol. In the absence of electron donor and electron carrier, oxyhaemoglobin and aniline give rise to the reversible production of 4-aminophenol.

Stabilization of pepsin on duolite for the continuous hydrolysis of bovine haemoglobin at pH2 and 40°C

Several methods of pepsin immobilization have been applied in order to achieve the continuous hydrolysis of a 2.5% haemoglobin solution at pH 2 and 40°C. Methods using glutaraldehyde were unsuccesful because of the unstability of the derived enzyme at low pH. Pepsin covalently bounded to a Duolite amine resin by a carbodiimide showed a half life of 15 days during the hydrolysis of haemoglobin in a column reactor. No enzyme activity was detected in the hydrolysates. No accumulation of haem in the column was noticed which could have limited long term studies by plugging the system. Modulation of the degree of hydrolysis was also performed by changing the feeding flow rate of the reactor.

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.

Comparative study of native and chemically modified chymotrypsin as monomers, soluble polymers and membranes

Abstract α-Chymotrypsin (E.C. 3.4.21.1.) has been immobilized in the form of monomers, soluble polymers and membranes with glutaraldehyde. Chemically modified monomers (CMMs) of α-chymotrypsin were separated chromatographically on Sephadex G-100 to verify that they had the same molecular weight as the native enzyme. In addition, electrophoretic separation was carried out to determine the modifications due to the chemical reaction between free amine fractions and glutaraldehyde. The Michaelis constant of the native enzyme, CMMs, the soluble polymers and membranes was measured. It is identical for the native enzyme and CMMs, and increases with the degree of polymerization for soluble polymers and membranes. Increasing stability was observed in the following order: native enzyme, CMMs, soluble polymers and the membrane at 50°C. These experiments permitted the study of the influence of chemical modifications on the enzyme kinetics.

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.