Hemoglobin Hydrolysis Research Articles

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 photodi.ode 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.

Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus

De Vicente, J. I., De Arriaga, D., Del Valle, P., Soler, J., and Eslava, A. P. 1996. Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus. Fungal Genetics and Biology20,115–124. An acid protease has been found in the culture broth ofPhycomyces blakesleeanusgrowing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease ofPhycomycesmigrated as a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A–Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70°C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50°C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-NO2)-Arg-Leu as the substrate, theKm,kcat, andVmaxvalues were 8.78 μM, 1.25 s−1, and 2.12 μmol min−1mg−1, respectively. The protease was insensitive to phenylmethylsulfonyl fluoride,O-phenanthroline,N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with aKivalue of 1.33 nM.The data suggest that this protease has properties of an aspartate-type proteinase.

Removal of Arg141 from the alpha chain of human hemoglobin by carboxypeptidases N and M.

Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (alpha-Arg141) of the alpha chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of alpha-Arg141 accelerated the dissociation rate of the tetramer deoxy-des-alpha-Arg141 hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-alpha-Arg141 hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-alpha-Arg141 hemoglobin had a higher oxygen affinity (P50, 5.51 mm Hg; control, 19.94 mm Hg [P50 is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P50, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des-alpha-Arg141 hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the alpha-Arg141 residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.

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.

Comparison of complex organic media for the cultivation of the temperature-sensitive mutant Tetrahymena thermophila SJ180

This is the first report on the mass cultivation of the mutant Tetrahymena thermophila SJ180; this strain has a temperature-sensitive food vacuole formation. The generation time, at both 28°C (permissive temperature) and 39°C (restrictive temperature) was determined in three commonly used complex media based, respectively, on proteose peptone (PPYS), skim milk (MYE), and hemoglobin hydrolysate (HB). The HB medium cost can be reduced by using a hemoglobin hydrolysate containing hemin residues (the decolorization step is thus avoided). The SJ180 strain grew in this medium only at 28°C, at which it formed food vacuoles. The HB medium, which is more stable in its amino acid composition, was improved by glucose addition; this resulted in high cell densities. By omitting the major nitrogen source in complex media, we demonstrated that at both 28°C and 39°C, the SJ180 strain could be cultivated in media composed simply of yeast extract. Moreover, if salt solutions were added to the yeast extract, the results were similar to those in the three complex media (PPYS, MYE, and HB). Finally, we developed a very simple medium composed of yeast extract and glucose, which resulted in a longer stationary phase and cell populations twice as high as that in the reference media. Consequently, this medium is very advantageous, because it reduces the subculture numbers in cell collection.

Production of Peptic Hemoglobin Hydrolyzates: Bitterness Demonstration and Characterization

Hemoglobin could be valorized in enzymatic hydrolysate form aimed at human and animal feeding. Thus, the hydrolysis of hemoglobin by pepsin was studied. A bitter taste in the hydrolysates was encountered, so different separation processes were developed to isolate the bitter fractions according to several characteristics. Therefore, ultrafiltration, reversed phase chromatography, and organic solvent extraction were used. Bitter peptides were concentrated in YM5 membrane (cutoff : 5000) permeate. They were eluted with 30-40% CH 3 CN from a C 18 bonded silica column and selectively extracted by 2-butanol. They were also adsorbed on a Superose 12 gel filtration column, and so they were represented by a chromatographic peak appearing after total column volume. Chromatography on Superose 12 could thus constitute an interesting analytical method for detecting bitterness in hydrolysates.

Functional expression of falcipain, a Plasmodium falciparum cysteine proteinase, supports its role as a malarial hemoglobinase.

Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin. To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems. After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase. Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin. Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases. Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides.

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.

Bitter peptide from hemoglobin hydrolysate: Isolation and characterization

Two separation methods, ultrafiltration and 2-butanol extraction, have shown that a peptide is the major agent responsible for bitterness in peptic hemoglobin hydrolysates. It was easily purified from these complex mixtures by specific hydrophobic adsorption on Superose 12, a gel-filtration column, which could constitute an original and interesting method for bitterness detection. The bitter peptide which corresponded to VV-hemorphin 7, the fragment 32–40 of the β chain of bovine hemoglobin, is first generated during proteolysis, then hydrolysed by pepsin. It exhibited a strong bitterness at 0.25 mM equivalent to 0.073 mM quinine sulfate or 21 mM caffeine.

Determination of hemoglobin adduct levels of the carcinogen 2,4-diaminotoluene using gas chromatography-electron impact positive-ion mass spectrometry.

A procedure to determine hemoglobin adduct yields resulting from exposure to the carcinogen 2,4-diaminotoluene (2,4-TDA) was developed using gas chromatography-electron impact positive-ion mass spectrometry. Liberated 2,4-TDA was quantified following alkaline hydrolysis of hemoglobin. Optimized derivatization of free 2,4-TDA with hepatafluorobutyric anhydride allowed detection of hemoglobin adduct levels as low as 5 ng/g Hb. Pure HFBA-2,4-TDA showed a linear dynamic range of 50 to 5000 pg. The quantitative extraction and recovery of liberated 2,4-TDA (ca. 100%) following hemoglobin hydrolysis allows accurate and precise determinations of adduct yields.

VV-hemorphin-7 and LVV-hemorphin-7 released during in vitro peptic hemoglobin hydrolysis are morphinomimetic peptides

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.

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.

Extraction of harp seal gastric proteases and their immobilization on chitin

Gastric proteases from seal stomach were isolated following homogenization, extraction, centrifugation and ammonium sulfate precipitation/fractionation. The crude seal gastric proteases (SGP) were stable in acid conditions with optimum stability at pH 3.0, but were unstable in the alkaline range. The crude SGP possessed excellent temperature adaptability with relative activities of 70 and 90% at 5 and 25 °C, respectively. The activity of crude SGPs was retained up to about 40 min incubation at 70 °C. The isolated SGP were immobilized on glutaraldehyde-treated chitin. The immobilized SGP exhibited optimum performance at pH 2.0, were most stable at pH 4.0 and had a 90 h half-life in a column with continuous operation for haemoglobin hydrolysis at room temperature. The native SGP clotted milk rapidly at pH 5.8 to 6.6; however, immobilized SGP had a lower milk-clotting activity.

Inhibition and Inhibition Kinetics of Angiotensin Converting Enzyme Activity by Hemorphins, Isolated from a Peptic Bovine Hemoglobin Hydrolysate

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.

Stability of a mineral membrane ultrafiltration reactor for peptide hydrolysis of hemoglobin

AbstractFactors involved in the activity decay of an ultrafiltration reactor during the hydrolysis of hemoglobin by pepsin were studied. Biochemical and hydrodynamic parameters were assessed. The product of hydrolysis showed no inhibitory effect. Autolysis and mechanical damage of pepsin were limited by the presence of hemoglobin. Activity decay was mainly a result of the leakage of pepsin through the membrane. Electrostatic charges carried by either hemoglobin or pepsin were involved in the permeation process.

Organization of digestion and preliminary characterization of salivary trypsin-like enzymes in a predaceous heteropteran, Zelus renardii

Salivary and intestinal proteolytic enzymes were studied in the predaceous heteropteran, Zelus renardii. Proteinases from Z. renardii's salivary gland complex (SGC), anterior midgut (AMG) and posterior midgut (PMG) were studied with hemoglobin, BApNA, BTNA, HPLA, HA and LPNA used as substrates. The SGC was shown to be an important source of general proteinase and endopeptidase activity, but exopeptidase activity (LPNA, HPLA and HA hydrolysis) was limited to the AMG and PMG. The AMG was also a source of considerable endopeptidase activity. Hemoglobin or BApNA hydrolysis occurred at a broad range of temperatures and pH with most activity observed at 35–45°C and at pH 7.0–8.5. The pH of the prey increased, as it was being pre-orally digested. The salivary trypsin-like enzyme had a molecular weight of approx. 27 kDa and eluted as two major activity peaks from an ion exchange column. BApNA hydrolysis was inhibited by soy proteinase inhibitor. Digestion, with respect to protein hydrolysis, is organized into an orderly and highly efficient process, differing greatly from that of hematophagous reduviids, beginning with pre-oral digestion of the prey's internal structures whose liquefaction permits ingestion and further digestive processing of prey within the predator's gut.

A Plasmodium vinckei cysteine proteinase shares unique features with its Plasmodium falciparum analogue

The gene encoding a cysteine proteinase of the murine parasite Plasmodium vinckei has been identified and characterized. The gene encodes a papain-family proteinase that shares unique features with a previously described P. falciparum cysteine proteinase. We hypothesize that both enzymes mediate the hydrolysis of hemoglobin, and perhaps other Plasmodium-specific functions.

Tobacco-Specific Nitrosamine Adducts: Studies in Laboratory Animals and Humans

This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers of the lung, esophagus, oral cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 200-1800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridyloxobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O6-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridyloxobutylation enhances both persistence of O6-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined.(ABSTRACT TRUNCATED AT 250 WORDS)

Tobacco-specific nitrosamine adducts: studies in laboratory animals and humans.

This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers of the lung, esophagus, oral cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 200-1800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridyloxobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O6-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridyloxobutylation enhances both persistence of O6-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined.(ABSTRACT TRUNCATED AT 250 WORDS)

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.