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Efficiency and selectivity of 30 and 150 kd inorganic ultrafiltration membranes (Techsep) toward tuna hemoglobin and myoglobin were studied. The influence of pH and ionic strength was investigated. Mass flow of myoglobin was higher at its isoelectric pH (8.6) and for low ionic strength (1.5 mM). This result was related to the absence of electrostatic repulsion between myoglobin and the surface of the dynamic membrane. The use of high ionic strength 0.15 M NaCl involved an apparent dimerisation of myoglobin and consequently a lower permeation through the membrane due to the molecular weight increase. The permeation and retention of hemoglobin did not agree with the effect of pH observed with myoglobin (best permeation at isoelectric pH) but followed the behavior of myoglobin. This was explained by a myoglobin concentration 10 times higher than hemoglobin concentration. The yield of retention selectivity was investigated. Selectivity of the membrane at pH 8.6 and 1.5 mM was favorable to myoglobin (increase of 40%) whereas a reversed selectivity was observed at pH 7.3, 0.15 M. (c) 1996 John Wiley & Sons, Inc.

A peptide with a bacterial-growth-stimulating activity was isolated from a bovine hemoglobin hydrolysate by reversed-phase high-performance liquid chromatography. Its primary structure and molecular mass, determined by amino acid analysis and fast-atom bombardment mass spectrometry, were identical to those of fragment 48-52 (Ser-Thr-Ala-Asp-Ala) of the beta chain of bovine hemoglobin. The microbiological tests in solid media demonstrated that this peptide exhibited a growth-stimulating activity on gram-negative bacteria.

The kinetics of appearance of hemorphins during peptic hydrolysis of bovine hemoglobin was investigated by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector. The degree of hydrolysis (DH) of hemoglobin by pepsin was determined and different defined DH of hydrolysates were obtained. The analysis of these hydrolysates by HPLC coupled with a photodiode array detector allowed us to identify and quantify the hemorphins in every hydrolysate and to determine the quantitative evolution of hemorphins as a function of DH. It indicated that hemoglobin was a direct precursor of LVV-hemorphin-5 and LVV-hemorphin-7. These peptides were demonstrated to be secondary substrates for pepsin to generate VV-hemorphin-5 and VV-hemorphin-7. Moreover, LVV-hemorphin-7 was more stable towards pepsin than LVV-hemorphin-5. The affinity of pepsin towards some peptidic bonds was also demonstrated.

Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D. Analysis of resulting peptides, by reversed-phase high-performance liquid chromatography (RP-HPLC), showed the release of a bioactive peptide, VV-hemorphin-7. When a carboxyl proteinase inhibitor such as pepstatin A was added, no hemorphin was generated. Our results clearly demonstrated that VV-hemorphin-7 generation was principally due to cathepsin D. This study allowed us to hypothesize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages.

The characterization of aromatic amino acid-containing peptides in biological fluids or protein hydrolysates is commonly achieved using classical size-exclusion (SE) and reversed-phase (RP) high-performance liquid chromatography (HPLC) coupled with direct ultraviolet (UV) spectrometry. Here, a non-destructive quantitative determination of aromatic amino acids in peptides is developed using second-order derivative spectra obtained during RP-HPLC coupled with photodiode array detection. In this method, the free aromatic amino acids were used as standards. Sensitivity and accuracy were verified using some peptides, including bioactive hemorphins. The method was applied to determine the amounts of hemorphins present in a complex peptic bovine hemoglobin hydrolysate.

Bovine globin has been hydrolysed by pepsin to different degrees of hydrolysis. Analysis of the hydrolysates, by reversed-phase high-performance liquid chromatography (RP-HPLC), shows the release of LVV- and VV-hemorphin-7. LVV-hemorphin-7 was the first generated, at a degree of hydrolysis (DH), as low as 4%. In contrast, VV-hemorphin-7 was produced later. Our study clearly shows that VV-hemorphin-7 is issued directly from LVV-hemorphin-7, since this later completely disappeared during hydrolysis. This work allows us to suggest a possible pathway for in vivo hemorphins appearance.

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.

Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.

Two hemorphins, peptides with opioid activity, have been isolated from a pepsin hydrolysate of bovine hemoglobin, 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. Two other peptides, 34-40 (hemorphin-7) and 34-41 (hemorphin-8) of the beta-chain of bovine hemoglobin, have been synthesized and studied. The opioid potency of these peptides, exhibited by the use of electrically stimulated muscle of isolated guinea pig ileum (GPI), were significant and comparable with some others previously described. Studies of opioid activities and primary structure of hemorphins led us to postulate the important role of arginine and phenylalanine in opioid potency.

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.

The inhibitory effect on angiotensin converting enzyme (ACE) activity by hemorphins isolated from an enzymatic bovine hemoglobin hydrolysate was reported. Inhibitory kinetics proved that inhibition mechanism was non-competitive. The stability of hemorphins towards ACE was studied.

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.

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.

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.

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.

Ten patients with chronic respiratory failure (PaO2 = 56,19 +/- 10,54; PaCO2 = 49,17 +/- 7,31 torr) were given intramuscular injections of tiapride, Changes in alveolar ventilation were assessed clinically and by monitoring blood gases. No adverse effects were recorded. PaO2 was similar before and one hour after tiapride. There was no hypoventilation but, on the contrary, a slight decrease in PaCO2 (49,17 +/- 7,3 and 47,10 +/- 7,3 torr) and a significant increase in pH (7,358 +/- 0,023 and 7,378 +/- 0,028; p less than 0,05). Tiapride was also given to ten patients before fiberoptic bronchoscopy. Endoscopy was well tolerated in eight patients, in six there was no anxiety, and in all normal vigilance was retained.

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.