I. Garreau

Opioid peptides derived from hemoglobin: Hemorphins

Investigation of hemoglobin peptic hydrolysate has revealed the presence of biologically active peptides with affinity for opioid receptors. Two peptides, VV-hemorphin-7 and LVV-hemorphin-7, were resolved by a combination of size exclusion and reversed phase HPLC. A new spectroscopic method based on the second order derivative spectra analysis of aromatic amino acids has been developed. This method allows qualitative and quantitative evaluation of hemorphins generated by peptic hemoglobin hydrolysis. Using this method, a kinetic study of hemorphins appearance has been undertaken. In this paper, we also evidenced the generation of VV-hemorphin-7 from globin by peritoneal macrophages. In regard to this result, the putative physiological role of hemorphins is discussed.

Hemorphins inhibit angiotensin IV binding and interact with aminopeptidase N

[125I]-Ang IV binding to rabbit collecting duct cell membranes was inhibited by hemorphins (H), a class of endogenous peptides obtained by hydrolysis of the beta chain of hemoglobin. The most potent competitors were those with a valine in their N-terminal part such as LVV-H7 and VV-H7 (IC50 = 1.3 nM) followed by VV-H8 and K6VV-H7 (5.1 nM). The same H, like Ang IV, interacted with aminopeptidase N (APN) as shown by their inhibitory effect (28-36%) on APN activity. HPLC analysis showed that only H with a N-terminal valine or leucine were hydrolyzed. Since H are detected in the body fluids, they are likely to act as endogenous competitors of Ang IV.

Reversed-phase high-performance liquid chromatography coupled with second-order derivative spectroscopy for the quantitation of aromatic amino acids in peptides : application to hemorphins

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.

Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins.

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin‐7‐related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.

Generation of VV-hemorphin-7 from globin by peritoneal macrophages

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), shown 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 hypothetize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages.

Hemorphin peptides are released from hemoglobin by cathepsin D. radioimmunoassay against the C-part of V-V-hemorphin-7: an alternative assay for the cathepsin D activity.

In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.

Identification of Hemorphins from Bovine Hemoglobin Hydrolysate: Application of UV Second Order Derivative Spectroscopy

Abstract Aromatic amino acids have very informative second order derivative spectra. Whereas they exhibit overlapping maxima between 250 and 300nm in the zero order spectra, thin minima are obtained in their second order derivative spectra. This feature allowed to develop a method to identify aromatic amino acids, but also to calculate the ratio between these amino acids in peptides and proteins. This method has been used successfully for the detection of hemorphins in a peptic bovine hemoglobin hydrolysate. The constant ratios between aromatic amino acids are an important characteristic of lots of bioactive peptides; the advantage of this spectral method is to be non-destructive for the identification of these amino acids espacially for tryptophan.

Separation of hemoglobin and myoglobin from yellowfin tuna red muscle by ultrafiltration: Effect of pH and ionic strength.

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.

Analytical Peptide Mapping of a Complex Yellowfin Tuna Myoglobin Peptic Hydrolysate by High Performance Liquid Chromatography

Abstract A method is described for a real identification of any peptides isolated from a complex peptic Yellowfin tuna red muscle myoglobin hydrolysate. A combination of size exclusion and reversed phase high performance liquid chromatography have proved to be a useful strategy for fractionation of such a mixture. This technique enable a large number of pure peptides from the total hydrolysate to be obtained. Peptides were identified and located on the known myoglobin sequence from their amino acid content determined by the Pico-Tag method and a second order derivative spectroscopic method. Location of the peptides allowed us to define effective cut sites of the porcine pepsin. The procedure described in this study will be useful for acquiring a better knowledge of such an hydrolysate.

Identification of hemorphins in a cathepsin D bovine hemoglobin hydrolysate by radioimmunoassay and photodiode array detections

Morphinomimetic peptides have been purified from hemoglobin enzymatic hydrolysates and a significant amount of evidence has been accumulated indicating that the generation of these peptides (hemorphins) might occur in vivo. In order to investigate their putative physiological role and processing from hemoglobin in vivo, two methods were developed: a specific radioimmunoassay and a UV spectra comparison analysis. These methods were applied to a cathepsin D bovine hemoglobin hydrolysate and allowed the detection of two hemorphin-7 peptides. This observation supports the putative implication of cathepsin D in the in vivo release of hemorphins. Among the two methods used in this study, the immunological approach exhibits higher sensitivity and represents a useful method to investigate the in vivo role and physiological processing of hemorphins.