Bovine Hemoglobin Hydrolysate Research Articles

Potential of Human Hemoglobin as a Source of Bioactive Peptides: Comparative Study of Enzymatic Hydrolysis with Bovine Hemoglobin and the Production of Active Peptide α137-141.

Cruor, the main component responsible for the red color of mammalian blood, contains 90% haemoglobin, a protein considered to be a rich source of bioactive peptides. The aim of the present study is to assess the potential of human hemoglobin as a source of bioactive peptides, compared with bovine hemoglobin, which has been extensively studied in recent years. More specifically, the study focused on the α137-141 fragment of bovine haemoglobin (TSKYR), a small (653 Da) hydrophilic antimicrobial peptide. In this work, the potential of human hemoglobin to contain bioactive peptides was first investigated in silico in comparison with bovine hemoglobin-derived peptides using bioinformatics tools. The blast results showed a high identity, 88% and 85% respectively, indicating a high similarity between the α and β chains. Peptide Cutter software was used to predict cleavage sites during peptide hydrolysis, revealing major conservation in the number and location of cleavage sites between the two species, while highlighting some differences. Some peptides were conserved, notably our target peptide (TSKYR), while others were specific to each species. Secondly, the two types of hemoglobin were subjected to similar enzymatic hydrolysis conditions (23 °C, pH 3.5), which showed that the hydrolysis of human hemoglobin followed the same reaction mechanism as the hydrolysis of bovine hemoglobin, the 'zipper' mechanism. Concerning the peptide of interest, α137-141, the RP-UPLC analyses showed that its identification was not affected by the increase in the initial substrate concentration. Its production was rapid, with more than 60% of the total α137-141 peptide production achieved in just 30 min of hydrolysis, reaching peak production at 3 h. Furthermore, increasing the substrate concentration from 1% to 10% (w/v) resulted in a proportional increase in α137-141 production, with a maximum concentration reaching 687.98 ± 75.77 mg·L-1, approximately ten-fold higher than that obtained with a 1% (w/v) concentration. Finally, the results of the UPLC-MS/MS analysis revealed the identification of 217 unique peptides in bovine hemoglobin hydrolysate and 189 unique peptides in human hemoglobin hydrolysate. Of these, 57 peptides were strictly common to both species. This revealed the presence of several bioactive peptides in both cattle and humans. Although some had been known previously, new bioactive peptides were discovered in human hemoglobin, such as four antibacterial peptides (α37-46 PTTKTYFPHF, α36-45 FPTTKTYFPH, α137-141 TSKYR, and α133-141 STVLTSKYR), three opioid peptides (α137-141 TSKYR,β31-40 LVVYPWTQRF,β32-40, VVYPWTQRF), an ACE inhibitor (β129-135 KVVAGVA), an anticancer agent (β33-39 VVYPWTQ), and an antioxidant (α137-141 TSKYR). To the best of our knowledge, these peptides have never been found in human hemoglobin before.

Ultrafiltration Fractionation of Bovine Hemoglobin Hydrolysates: Prediction of Separation Performances for Optimal Enrichment in Antimicrobial Peptide.

Hydrolysis of bovine hemoglobin (bHb), the main constituent of bovine cruor by-product, releases a natural antimicrobial peptide (NKT) which could present a major interest for food safety. To enrich this, tangential ultrafiltration can be implemented, but ultrafiltration conditions are mainly empirically established. In this context, the application of a simulation method for predicting the NKT yield and enrichment was investigated. Ultrafiltration performances were studied for decolored bHb hydrolysates at different degrees of hydrolysis (DH; 3%, 5%, 10% and 18%) and colored hydrolysates (3% and 5% DH) with 1 and 3 kg·mol−1 regenerated cellulose membranes. The simulation method helped to identify the most promising hydrolysate (in terms of NKT enrichment, yield and productivity) as the 3% DH colored hydrolysate, and UF conditions (volumetric reduction factor of 5 and 3 with 1 and 3 kg·mol−1 membrane, respectively) for higher antimicrobial recovery. A maximal enrichment factor of about 29 and NKT purity of 70% in permeate were observed. The results showed that the antimicrobial activity was in relation with the process selectivity and NKT purity. Finally, this reliable method, applied for predicting the ultrafiltration performances to enrich peptides of interest, is part of a global approach to rationally valorize protein resources from various by-products.

Slaughterhouse By-Product Valorization: Hydrolysis Degree Modification for Higher Antimicrobial Recovery by Electroseparation

In the actual context of food safety and circular economy, the separation of the α137–141 antimicrobial peptide (TSKYR, 653 Da) coming from the hydrolysis of bovine hemoglobin, a non-valorized slaughterhouse by-product, and its reuse as a preservative agent for food products during storage would be of major interest. However, depending on the degree of hydrolysis (DH) of the bovine hemoglobin, the TSKYR peptide environment into the hydrolysate will be different and would impact its recovery yield by electrodialysis with ultrafiltration membranes (EDUF), a hybrid and eco-friendly technology. In this context, five DHs (3, 5, 10, 13 and 18%) were investigated to study the influence of the peptide populations on the α137–141 selective separation by EDUF. It appeared from these results that the most appropriate DH was of 5% since a lower population of peptides between 500 and 1000 Da was present and the subunits of hemoglobin were already digested. Hence, an enrichment factor of about 13 folds for the α137–141 was obtained in comparison with the initial hydrolysate. At our knowledge, it was the first time that the effect of the hydrolysis degree of a protein is demonstrated to impact preferentially the separation of a specific peptide by EDUF. Graphic

Studies on Chromatographic Fractioning by Cations Exchangers of a Bovine Hemoglobin Hydrolysate

A peptidic hydrolysate has been obtained through hydrolysis of bovine hemoglobin using pepsin. The fractioning of the hydrolysate was performed on a column packed with CM-Sepharose Fast Flow. The hydrolysate and each fraction was filtered and then injected into a HPLC system equipped with a Vydak C4 reverse phase column (0.46 x 25 cm), suitable for the chromatographic separation of large peptides with 20 to 30 amino acids. The detection was done using mass spectrometry, and the retention time, size and distribution of the peptides were determined.

Identification and Inhibitory Mechanism of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Bovine Hemoglobin.

Angiotensin I-converting enzyme (ACE, EC.3.4.15.1) inhibitory peptide is an efficacious therapy for hypertension. In this study, four dipeptides, TY, FD, FL and FG, were identified from the desalted fraction of bovine hemoglobin hydrolysate, obtained by in vitro simulated gastrointestinal digestion, via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The IC50 value of TY and FL are 96.43 ± 6.17 and 290.66 ± 57.92μM, respectively. The result of molecular docking indicated that TY occupied the ACE subsite S1 and S1' with a lowest estimated binding energy of -9.96Kcal/mol, while FL occupied the subsite S5 with a lowest estimated binding energy of -9.37Kcal/mol. The subsite S1' and S2' are closer to the ACE active center (Zn2+) than S5, and the lowest estimated binding energy of TY is lower than that of FL. This work provided new ACE-inhibitory peptides derived from bovine hemoglobin hydrolysate and explained their inhibitory mechanism.

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum.

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.

Optimization of Peptide Separation from Complex Peptide Mixture in a Foaming-Draining System

The physico-chemical environment of the separation, applied on a complex peptide hydrolysate obtained from the hydrolysis of bovine hemoglobin by pepsin, to recover an antimicrobial peptide fraction was shown to influence its recovery efficiency and selectivity. The objective of the present work was to evaluate experimental conditions that maximize the adsorption capacity of the antimicrobial peptide fraction on liquid/air interface through the use of an experimental design methodology. A first step comprised a study on several factors thanks to a screening matrix to acquire basic knowledges on these variables and their influence on the extraction selectivity. Hydrolysate and salt concentrations, pH of both the hydrolysate and draining solutions were established as the four main factors. Then a composite matrix was studied to identify with response surface modelling the optimum conditions for fractionation.

α 67-106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides

Protein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on bovine hemoglobin hydrolysate obtained with pig pepsin in the presence of 30% ethanol. This hydrolysate was fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) into 12 major fractions (F1–F12). All fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize and identify the peptides in these fractions. This hydrolysis permitted to generate a new serial of bioactive peptides with both antimicrobial and ACE inhibitory activities. Identified peptides were TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL, LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL, KLLSHSL, and LLSHSL corresponding respectively to the 67-106, 73-105, 99-105, and 100-105 fragments of the α chain of bovine hemoglobin. They were the first found from bovine hemoglobin. These purified peptides have an antibacterial activity against four bacteria strains: Kocuria luteus A270, Listeria innocua, Escherichia coli, and Staphylococcus aureus with a MIC between 187.1 and 35.2 μM. On the other hand, these peptides displayed at the same time ACE inhibitory activity with an IC50 range from 42.55 to 1,095 μM.

Advancement of foam separation of bioactive peptides using an aeration column with a bubbling-draining method

Peptidic hydrolysates with different degrees of hydrolysis (3, 15 and 23%) has been obtained by hydrolysis of bovine haemoglobin by pepsin. RP-HPLC analysis of these hydrolysates coupled with mass spectroscopy has given information about the retention time and the peptidic size distribution. Pendant-drop tensiometry of aqueous-buffer solutions versus concentration points out differences in the curves superficial tension versus concentration between starting haemoglobin and hydrolysates. The weight percentage of hydrolysates in the bulk C B max for which stable foam can be obtained has been determined. Foam fractionation was studied by a bubbling-draining method. Collected top foam was analyzed by RP-HPLC and compared to the initial hydrolysate. No stable foam has been obtained with hydrolysate (DH = 23%) and no interesting fractionation can be obtained with hydrolysate (DH = 15%). However, a peptidic fraction (3500–7000 Da), having an antimicrobial activity, with a 5% extraction yield can be separated from hydrolysate (DH = 3%), using a forced draining volume of 100 or 150 mL.

Identification and Release Kinetics of Peptides from the Process of Peptic Hydrolysis of Bovine Hemoglobin by LC‐ESI‐MS/MS

Bovine hemoglobin was hydrolyzed with pepsin in a batch stirred tank reactor; the resulting peptides were identified, in a time dependent and comprehensive manner, using reversed phase‐high performance liquid chromatography (RP‐HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI‐MS/MS) by means of database searching. Peptic digestion of bovine hemoglobin yields more hydrophobic peptides at a low degree of hydrolysis, and more hydrophilic peptides at a later stage of hydrolysis. The release kinetics of the bioactive peptides was also followed based on the RP‐HPLC profiles. In addition, the behavior of peptic digestion of hemoglobin α and β chains was compared in terms of profiling the identified peptides. Thirty‐two peptides were recovered by a process of hydrolysis from the α chain of the peptide; whereas the corresponding result for the β chain was 19 peptides with around 67% sequence coverage. The main factor responsible for non‐peptic susceptibility of the central region of the β chain was their relatively higher hydrophilicity.

Influence of pH on the Appearance of Active Peptides in the Course of Peptic Hydrolysis of Bovine Haemoglobin

Influence of pH on the appearance of active peptides in peptic hydrolysis of bovine haemoglobin was studied in a homogenous phase system. Six active peptides were studied: three hemorphins: LVVH‐7 (β 31–40), VVH‐7 (β 32–40), VVH‐4 (β 32–37), one bradykinin‐potentiating peptide (α 110–125), one antibacterial peptide (α 1–23), and neokyotorphin (α 137–141). The influence of pH was investigated in the course of the hydrolysis of haemoglobin by pepsin at 23°C in acetate buffer at pH 3.5, pH 4.5, and pH 5.5. The hydrolysis of haemoglobin was studied in the presence or absence of urea. The haemoglobin hydrolysis at pH 4.5 is taken as a reference. Two different mechanisms of hydrolysis were observed: “one by one” for native haemoglobin hydrolysis at pH 4.5 and 5.5, and “zipper” for denatured haemoglobin at pH 3.5, pH 4.5, and pH 5.5, and native haemoglobin at pH 3.5. Whatever the pH and medium, a selectivity change by the pepsin was noticed. In the presence of urea, there are two phenomena: some peptides are preferentially produced at pH 3.5 and other peptides at pH 5.5, which seems to favour one particular site of pepsin that is cut. In the absence of urea, these active peptides reached a higher concentration at pH 3.5. In order to prepare these six active peptides, it is suitable to hydrolyse haemoglobin in the absence of urea at pH 3.5 (this pH denatures haemoglobin) where a “zipper” mechanism is obtained, and the peptide quantity is more significant at pH 3.5 than at pH 4.5.

On-Line Mid-Infrared Spectroscopic Data and Chemometrics for the Monitoring of an Enzymatic Hydrolysis

Multivariate models have been designed in order to be implemented at pilot plant scale. They allow the prediction of the degree of the enzymatic hydrolysis of bovine hemoglobin from on-line mid-infrared (MIR) attenuated total reflection (ATR) measurements. The procedure is very safe and practical, but the spectral information is unfortunately difficult to use. First, remote MIR optical fiber measurements induce a significant energy loss. Secondly, the spectra of proteins in aqueous solutions generally consist of broad and overlapping signals. Principal component analysis (PCA) of the full data set reveals some variations between the different batches as well as definite deviations from the ideal linear situation. This suggests the use of artificial neural networks (ANNs), which have the useful qualities of generalization ability and robustness. Preprocessing of the spectral data (including PCA and wavelet transforms) have been tested. In addition, the reliability of the calibration is ensured by the interpretation of the spectroscopic information. This permits the construction of very parsimonious models and contributes to greater confidence in the analytical tool built for process monitoring. Finally, the estimations of the prediction error are demonstrated to be acceptable. With an unknown proteolysis batch, this error is found to be lower than 0.4 for the degree of hydrolysis covering the range 0 to 8.7.

Improvement of Staphylococcus aureus-V8-protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides.

Hydrolysis of bovine haemoglobin by the V8 protease from Staphylococcus aureus (EC 3.4.21.19) was studied in the presence of SDS in a homogeneous-phase and in a solid-phase system. In both cases, hydrolyses were performed at 37 degrees C, in 50 mM phosphate buffer, pH 6.0, containing 0.1% SDS. Solid-phase hydrolyses were carried out with haemoglobin adsorbed on a negatively charged hydrophobic support, namely Amberlyst 15Wet (Rohm and Haas). The peptides were isolated from the hydrolysates by reverse-phase HPLC and analysed for their amino acid composition on a Waters Pico-Tag column, confirmed by second-order derivative spectrometry or by MS. A peptide map of the hydrolysates was drawn up, and numerous new cleavages in haemoglobin chains were observed, especially after Asp. This study showed that SDS permitted a dramatic improvement in the hydrolysis of whole haemoglobin by V8 protease in both homogeneous-phase and solid phase systems after adsorption of haemoglobin on to an anionic support. Moreover, in the heterogeneous phase, all the theoretical cleavage sites of V8 protease, Asp as well as Glu bonds, were hydrolysed, except for four sites which were resistant owing to strong interactions with the support. These results led to us obtain two haemopoietic peptides, namely peptide alpha (Leu(76)-Pro-Gly-Ala-Leu-Ser-Glu(82)) and peptide beta (Lys(94)-Leu-His-Val-Asp-Pro-Glu(100)). These active peptides have never before been prepared from bovine haemoglobin, and they may have great potentialities in biotechnology.

Solvent effect on kinetics of appearance of neokyotorphin, VV-haemorphin-4 and a bradykinin-potentiating peptide in the course of peptic hydrolysis of bovine haemoglobin.

The kinetics of appearance of bioactive peptides (neokyotorphin, VV-haemorphin-4 and a bradykinin-potentiating peptide) were investigated in the course of the hydrolysis of haemoglobin by pepsin at 23 degrees C in 0.1 M sodium acetate buffer at pH 4.5. Isolation of these peptides was performed in one step by C(4) reversed-phase HPLC. We have shown that neokyotorphin, and VV-haemorphin-4 are two final peptides and that the bradykinin-potentiating peptide is an intermediate peptide. At pH 4. 5, LVV-haemorphin-7, VV-haemorphin-7 and VV-haemorphin-4 appeared successively in the course of the peptic hydrolysis. At pH 2, the optimum pH of the pepsin hydrolysis, VV-haemorphin-4 was not detected. The effect of the composition of the solvent on the peptic hydrolysis was studied to improve the preparation of these active peptides. The kinetics of appearance of these peptides were compared in the presence of 20% (v/v) ethanol, a stabilizing solvent of haemoglobin and in the presence of urea, a denaturant agent. The best conditions for preparing neokyotorphin and VV-haemorphin-4 were to achieve the peptic reaction in the buffer alone or in the presence of urea at a high degree of hydrolysis. For the preparation of the bradykinin-potentiating peptide, the hydrolysis of haemoglobin in the urea-containing buffer at a moderate degree of hydrolysis of 10% was suitable.

Hydrolysis of haemoglobin surveyed by infrared spectroscopy: I. solvent effect on the secondary structure of haemoglobin

The hydrolysis of bovine haemoglobin in an acetic acid/sodium acetate buffer enables to produce peptides of major importance in biomedical research. The global objective is to survey this reaction by infrared spectroscopy. This article concerns the first step: the evaluation by spectroscopy of the effect on protein secondary structure of the addition of ethanol in the buffer.Conformational changes are related to solvent–protein interactions as the protein folding is driven by the entropy of removing hydrophobic groups from contact with the solvent. Therefore, the stability of haemoglobin in an ethanol–water mixture results in a competition between the water structure, which is strengthened by the presence of the alcohol, and the solubility of hydrophobic residues. Previous non infrared experiments, based on mass spectrometry for example, have been reported for the investigation of the denaturation of haemoglobin by an organic solvent.The use of vibrational spectroscopy for protein secondary structure determination has proved its efficiency. We focus here on the study of the denaturing of haemoglobin in a water medium by addition of ethanol. As our investigations deal with very low concentrated haemoglobin, we apply a technique that uses films dried from dilute solution. Although it is not generally accepted that the protein conformation is retained when the solvent evaporates off, we validate this method comparing some results to previous infrared study made at upper concentrations with liquid sampling. We observe that Fourier Transform Infrared (FTIR) Spectroscopy, combined with few mathematical treatments, permits to estimate that haemoglobin remains in a native form unless a concentration of more or less 20% of ethanol is reached. For greater values modifications are perceptible on the infrared spectra.

Influence of molecular interactions on ultrafiltration of a bovine hemoglobin hydrolysate with an organic membrane

Inter-molecular interactions involved in the hydrolysate were studied in order to explain heme and peptide high retentions observed during the ultrafiltration of a bovine hemoglobin peptidic hydrolysate with a 10 kDa modified polyethersulfone membrane. Physico-chemical properties of the peptidic fractions of the retentate and of the permeate were characterized by UV/vis spectroscopy, SDS-PAGE electrophoresis, size-exclusion chromatography, reversed phase HPLC, hydrophobic interaction chromatography on hemin agarose, precipitation with sodium chloride and amino acid compositions. Two populations of peptides were revealed in the hydrolysate: one forming high-molecular weight hydrophobic associations retained by the membrane, and another more hydrophilic, giving no associations and freely passing through the membrane. Contrary to the peptides of the permeate, peptides retained by the membrane had a high affinity and a large binding capacity for the heme. Heme as polymers is mainly linked by hydrophobic interactions with peptide associations to form large heme–peptide aggregates. These results suggest that high rejections of heme and peptide, often reported in the literature, during ultrafiltration of hemoglobin hydrolysates, could be largely explained by these associations.

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.

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

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.

Investigation of inhibition angiotensin-converting enzyme (ACE) activity and opioid activity of two hemorphins, LVV-hemorphin-5 and VV-hemorphin-5, isolated from a defined peptic hydrolysate of bovine hemoglobin

Two peptides, LVV-hemorphin-5 and VV-hemorphin-5, were isolated from a defined peptic bovine hemoglobin hydrolysate by reversed-phase HPLC. These peptides were identified as 31–38 and 32–38 fragments of β chain of bovine hemoglobin. Their inhibitory activity towards angiotensin-converting enzyme and opioid potency were determined. Since their amino acid sequences show close homology with spinorphin, which is found in human cerebrospinal fluid and in the bovine spinal cord, the possible physiological role in vivo of these peptides was discussed.

Characterization of kininogenase activity of an acidic proteinase isolated from human kidney

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase HPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 micrograms MLBK equivalent mg-1.min-1 at pH 3.5 and 2.15 micrograms MLBK equivalent mg-1.min-1 at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp++ + was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinectic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K(m) = 0.69 +/- 0.08 microM; Kcat = 0.052 +/- 0.0095 s-1; Kcat/K(m) = 0.075 +/- 0.005 microM-1.s-1) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K(m) = 1.56 +/- 0.16 microM; Kcat = 0.0048 +/- 0.0001 s-1; Kcat/K(m) = 0.003 +/- 0.0003 microM-1.s-1). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. The results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.