Qiuyu Zhao

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.

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.

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.

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.

Kinetics of appearance of four hemorphins from bovine hemoglobin peptic hydrolysates by HPLC coupled with photodiode array detection.

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.

A Rapid Detection and Identification of Hemorphins Released from Bovine Hemoglobin Enzymatic Hydrolysis by Use of HPLC Coupled with Photodiode Array Detector

Abstract Identification of hemorphins issued from a complex hemoglobin enzymatic hydrolysate was carried out by UV-spectra comparison. Two hemorphins, VV-hemorphin-7 and LVV-hemorphin-7, were detected in a single step by the use of HPLC coupled with photodiode array detector. This technique greatly simplified the the multistage identification and purification strategy. This method could also be efficiently applied to the identification of peptides containing aromatic amino acids.

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.

Peptic Peptide Mapping by HPLC, on Line With Photodiode Array Detection, of a Hemoglobin Hydrolysate Produced at Pilot-Plant Scale from an Ultrafiltration Process

Abstract The analysis of a peptic bovine hemoglobin hydrolysate, produced at pilot plant scale, was carried out using two techniques: SE-HPLC and RP-HPLC. Analysis of amino acid composition, second-order derivative spectrometry and FAB mass spectrometry of isolated peptides allowed us to determine the exact positions of these peptides in the sequence of bovine hemoglobin. This, consequently, gave rise to a peptidic map of the hydrolysate. It also revealed, at the same time, some biologically active peptides in the hydrolysate. This information should find use in the potential future application of enzymatic bovine hemoglobin hydrolysate.

Analysis of peptides from bovine hemoglobin and tuna myoglobin enzymatic hydrolysate: use of HPLC with on-line second-order derivative spectroscopy for the characterization of biologically active peptides

Abstract The Chromatographic resolution of bovine hemoglobin and tuna myoglobin peptic hydrolysates was performed by use of successive SE-and RP-HPLC in order to obtain pure peptides. Analysis of their amino acid composition and second order derivative spectroscopy allowed us to determine the accurate identity of these peptides and consequently to locate them in both protein sequences. Some biologically active peptides generated from hemoglobin were also studied by these methods to investigate the kinetics of their appearance during peptic hydrolysis.