Thermolysin Hydrolysis Research Articles

Isolation, purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

SummaryThe angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC50 = 0.86 μm) and IEKPP (IC50 = 1.2 μm) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.

Blood pressure lowering effect of a pea protein hydrolysate in hypertensive rats and humans.

The blood pressure lowering effect of a pea protein hydrolysate (PPH) that contained <3 kDa peptides, isolated by membrane ultrafiltration from the thermolysin digest of pea protein isolate (PPI), was examined using different rat models of hypertension as well as hypertensive human subjects. The PPH showed weak in vitro activities against renin and angiotensin converting enzyme (ACE) with inhibitory activities of 17 and 19%, respectively, at 1 mg/mL test concentration. Oral administration of the PPH to spontaneously hypertensive rats (SHR) at doses of 100 and 200 mg/kg body weight led to a lowering of hourly systolic blood pressure (SBP), with a maximum reduction of 19 mmHg at 4 h. In contrast, orally administered unhydrolyzed PPI had no blood pressure reducing effect in SHR, suggesting that thermolysin hydrolysis may have been responsible for releasing bioactive peptides from the native protein. Oral administration of the PPH to the Han:SPRD-cy rat (a model of chronic kidney disease) over an 8-week period led to 29 and 25 mmHg reductions in SBP and diastolic blood pressure, respectively. The PPH-fed rats had lower plasma levels of angiotensin II, the major vasopressor involved in development of hypertension, but there was no effect on plasma activity or renal mRNA levels of ACE. However, renal expression of renin mRNA levels was reduced by approximately 50% in the PPH-fed rats, suggesting that reduced renin may be responsible for the reduced levels of angiotensin II. In a 3-week randomized double blind placebo-controlled crossover human intervention trial (7 volunteers), significant (p<0.05) reductions (over placebo) in SBP of 5 and 6 mmHg were obtained in the second and third weeks, respectively, for the PPH group. Therefore, thermolysin derived bioactive peptides from PPH reduced blood pressure in hypertensive rats and human subjects, likely via effects on the renal angiotensin system.

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC50=0.033mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC50=0.45μM) at 791.155m/z and NGTWFEPP (IC50=0.63μM) at 1085.841m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.

Angiotensin I-Converting Enzyme Inhibitory Activity of Gelatin Hydrolysates and Identification of Bioactive Peptides

In this project we report on the angiotensin I-converting enzyme (ACE)-inhibitory activity of a bovine gelatin hydrolysate (Bh2) that was submitted to further hydrolysis by different enzymes. The thermolysin hydrolysate (Bh2t) showed the highest in vitro ACE inhibitory activity, and interestingly a marked in vivo blood pressure-lowering effect was demonstrated in spontaneously hypertensive rats (SHR). In contrast, Bh2 showed no effect in SHR, confirming the need for the extra thermolysin hydrolysis. Hence, an angiotensin I-evoked contractile response in isolated rat aortic rings was inhibited by Bh2t, but not by Bh2, suggesting ACE inhibition as the underlying antihypertensive mechanism for Bh2t. Using mass spectrometry, seven small peptides, AG, AGP, VGP, PY, QY, DY and IY or LY or HO-PY were identified in Bh2t. As these peptides showed ACE inhibitory activity and were more prominent in Bh2t than in Bh2, the current data provide evidence that these contribute to the antihypertensive effect of Bh2t.

Effect of sonication on thermolysin hydrolysis of ovotransferrin

The effect of sonication on proteolytic hydrolysis of ovotransferrin and bioactivities of the hydrolysates were investigated, and the large peptide fragments left in the hydrolysate were characterised. The results showed that sonication could increase the reactive sulphydryl groups in 5% ovotransferrin solution by 50%, although there were no improvements in the overall degree of hydrolysis. Furthermore, SDS–PAGE and reverse-phase HPLC profiles did not show difference of the treated samples. Angiotensin-converting enzyme (ACE) inhibitory activity, but not antioxidant activity, of the thermolysin hydrolysate was improved in a dose dependent manner at prolonged sonication time. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) analysis revealed that thermolysin hydrolysate was composed of 926 peptides, of 99% having masses lower than 10 kDa and of 1% larger than 10 kDa derived from both N- and C-lobes of ovotransferrin.

Amino acid composition and antioxidant properties of pea seed ( Pisum sativum L.) enzymatic protein hydrolysate fractions.

The amino acid composition and antioxidant activities of peptide fractions obtained from HPLC separation of a pea protein hydrolysate (PPH) were studied. Thermolysin hydrolysis of pea protein isolate and ultrafiltration (3 kDa molecular weight cutoff membrane) yielded a PPH that was separated into five fractions (F1-F5) on a C(18) reverse phase HPLC column. The fractions that eluted later from the column (F3-F5) contained higher contents hydrophobic and aromatic amino acids when compared to fractions that eluted early or the original PPH. Fractions F3-F5 also exhibited the strongest radical scavenging and metal chelating activities; however, hydrophobic character did not seem to contribute to reducing power of the peptides. In comparison to glutathione, the peptide fractions had significantly higher (p < 0.05) ability to inhibit linoleic acid oxidation and chelate metals. In contrast, glutathione had significantly higher (p < 0.05) free radical scavenging properties than the peptide fractions.

Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions

Enzymatic hydrolysates from flaxseed protein were investigated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. Pepsin, ficin, trypsin, papain, thermolysin, pancreatin and Alcalase were used to hydrolyze flaxseed proteins followed by fractionation using ultrafiltration to isolate low-molecular-weight peptides, and separation of the Alcalase hydrolysate into cationic peptide fractions. Using N-(3-[2-furyl]acryloyl)-phenylalanylglycylglycine as substrate, the protein hydrolysates showed a concentration-dependent ACE inhibition (IC 50, 0.0275–0.151 mg/ml) with thermolysin hydrolysate and Alcalase cationic peptide fraction I (FI) showing the most potent activity. Flaxseed peptide fractions also showed no or moderate inhibitory activities against human recombinant renin (IC 50, 1.22–2.81 mg/ml). Kinetics studies showed that the thermolysin hydrolysate and FI exhibited mixed-type pattern of ACE inhibition whereas cationic peptide fraction II inhibited renin in uncompetitive fashion. These results show that the protein components of flaxseed meal possess peptide amino acid sequences that can be exploited as potential food sources of anti-hypertensive agents.

Comparison of ACE inhibitory and DPPH radical scavenging activities of fish muscle hydrolysates

To utilise Atlantic salmon, Coho salmon, Alaska pollack, and southern blue whiting as components of neutraceutical food and to clarify the potential physiological function of those fishes, their muscles were hydrolysed with pepsin, pancreatin or thermolysin. Methanolic extracts of fish muscle and their hydrolysates were prepared for analysis of angiotensin converting enzyme (ACE) inhibitory and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. Pepsin hydrolysates of all the fish samples did not increase degree of hydrolysis, extractive nitrogen content and bioactivities. Pancreatin and thermolysin improved the ACE inhibitory activity, and the activity was expressed following the peak of size exclusion chromatography (SEC). The DPPH radical scavenging activity was increased following pancreatin and thermolysin hydrolysis, but this activity was not related to the SEC result. Except for the lower DPPH radical scavenging activity of Alaska pollack pancreatin hydrolysate than those of the others, there was no significant difference in the ACE inhibitory and DPPH radical scavenging activities by fish species.

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate, enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC 50 values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL −1, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.

Isolation of Influenza Virus A Hemagglutinin C-Terminal Domain by Hemagglutinin Proteolysis in Octylglucoside Micelles

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

Effect of calcium on thermolysin hydrolysis of β-casein tryptic peptides

Bovine β -casein was hydrolyzed with trypsin in the presence of 2 m m NaCl, the hydrolysate was centrifuged and the supernatant was ultrafiltered on a 10-kDa membrane. Under these conditions, mainly the peptides β -CN 1–25 and β -CN 33–97 A 1 and A 2 were produced as determined by RP-HPLC and IES-MS analyses. This hydrolysate was further submitted to thermolysin hydrolysis in the presence of NaCl (2 m m ) or CaCl 2 (0.2 and 2 m m ). The results revealed accelerated hydrolysis by thermolysin of the peptide β -CN 1–25 in the presence of calcium ions (2 m m ), while the degradation of the peptide β -CN 33–97 A 1 and A 2 was affected by the presence of neither NaCl nor CaCl 2 . The effect of calcium on the rate of proteolysis of the peptide β -CN 1–25 might be related to the binding of calcium to the phosphoseryl cluster, inducing structural changes in the peptide, and the affinity of thermolysin for specific peptidic bonds, in particular, for the peptidic bonds Ser 22 –Ile 23 and Glu 11 –Ile 12 . Binding of calcium ions to the phosphoseryl residues of proteins could provide a means of controlling the release of specific peptide sequences during proteolysis.

Isolation of HIV-1 Pretense Inhibiting Peptide from Thermolysin Hydrolysate of Manila Clam Proteins

A peptide inhibiting HIV-1 protease was isolated from the hydrolysate of manila clam (Ruditapes philippinarum) proteins digested with thermolysin. The peptide was purified by using membrane filtration, gel permeation chromatography, ion exchange chromatography, and reverse phase HPLC. The amino acid sequence of the peptide was determined to be Ile-Tyr-Glu-Gly. This tetrapeptide sequence exists in some proteins of Physarum polycephalum and Mycobacterium smegmatis. Chemically synthesized Ile-Tyr-Glu-Gly showed the IC_(50) value of 22.3 μM.

바지락 단백질 Thermolysin 가수분해물의 Angiotensin Converting Enzyme 저해 Peptide의 특성

바지락 단백질의 thermolysin 가수분해물로부터 분자량 한계범위가 10,000 da인 한외여과막을 통과한 저분자량의 물질을 Sephadex LH-20 column을 이용한 겔 크로마토그래피에서 ACE에 대하여 저해 활성을 가지는 3개의 획분을 분취하여 SP-Toyopearl 650S column과 SuperQ-Toyopearl 650S column을 이용한 이온 교환 크로마토그래피에 의하여 4개의 활성 획분을 얻었다. 이 중 가장 높은 절해 활성을 나타내는 획분의 아미노산 조성은 alanine, leucine, isoleucine 및 threonine의 함량이 많았고, 활성 발현에 있어 C 말단 아미노산 잔기로서 중요한 역할을 하는 proline의 함량도 <TEX>$3.7\%$</TEX>%인 것으로 나타났다. ACE 저해 활성은 <TEX>$IC_{50}$</TEX> 값이 0.748 <TEX>$\mu$</TEX>g이었다. The peptides inhibiting angiotensin converting enzyme (ACE) were isolated from the hydrolysate of manila clam (Ruditapes philippinamm) proteins prepared with thermolysin. The thermolysin hydrolysate was pretreated with membrane filter (MW cut-off 10,000) to obtain the peptide fraction with ACE inhibition. The crude peptides were applied to a Sephadex LH-20 column and eluted with <TEX>$30\%$</TEX> methanol. The three active fractions (A, B and C) were collected and concentrated, and then applied to a SP-Toyopearl 650S column equilibrated with distilled water and was eluted with a linear gradient of NaCl concentration (0 to 1 M). The four active fractions (A-1, A-2, B-1 and C-1) were collected and concentrated, and then applied to a SuperQ-Toyopearl 650S column equilibrated with distilled water and was eluted with a linear gradient of NaCl concentration (0 to 1 M). The maximum inhibitory activity was observed in the fraction B-1Q showed the IC_{50} values of 0.748 <TEX>$\mu$</TEX>g. The abundant amino acids obtained from active fraction B-1Q were leucine, isoleucine, alanine and threonine.

Antihypertensive Activities of Peptides Derived from Porcine Skeletal Muscle Myosin in Spontaneously Hypertensive Rats

ABSTRACT: Antihypertensive activities derived from porcine skeletal muscle proteins were investigated. Thermolysin hydrolysates of porcine muscle water‐insoluble proteins demonstrated antihypertensive activities in spontaneously hypertensive rats when administrated in single oral doses. Hydrolysates of porcine myosin and peptides (Met‐Asn‐Pro‐Pro‐Lys, Ile‐Thr‐Thr‐Asn‐Pro, Met‐Asn‐Pro, Pro‐Pro‐Lys) with parts of the sequence of myosin showed antihypertensive activities. This is the first report of antihypertensive activities of peptides derived from muscle proteins of domestic animals. The hydrolysates of porcine muscle protein and their corresponding bioactive peptides might be utilized for physiologically functional foods.

Isolation of HIV-1 Protease-Inhibiting Peptides from Thermolysin Hydrolysate of Oyster Proteins

The peptides inhibiting HIV-1 protease were isolated from the hydrolysate of oyster (Crassostrea gigas) proteins prepared with thermolysin. The amino acid sequences of the peptides were determined as Leu-Leu-Glu-Tyr-Ser-Ile and Leu-Leu-Glu-Tyr-Ser-Leu. These sequences exist in some proteins of variola major virus or human cytomegalovirus. Chemically synthesized Leu-Leu-Glu-Tyr-Ser-Ile and Leu-Leu-Glu-Tyr-Ser-Leu showed IC50values of 20 and 15 nM, respectively, and behaved as competitive inhibitors for HIV-1 protease withKivalues of 13 and 10 nM, respectively. These peptides were more potent as an HIV-1 protease inhibitor than pepstatin A.

Angiotensin I-converting enzyme inhibitory activities of synthetic peptides related to the tandem repeated sequence of a maize endosperm protein.

Peptides that exist in the tandem repeated region formed by six units of Pro-Pro-Pro-Val-His-Leu in a maize endosperm protein γ-zein were chemically synthesized, and the angiotensin I-converting enzyme inhibitory activities of these peptides were investigated. Synthetic Val-His-Leu-Pro-Pro-Pro inhibited the enzyme (IC50 = 200 jim). Other synthetic fragment peptides, Val-His-Leu-Pro-Pro (IC50 = 18 μΜ) and Leu-Pro-Pro (IC50 = 9.6 μύ) inhibited the enzyme more potently. Then the native hexapeptide Val-His-Leu-Pro-Pro-Pro was purified from a thermolysin hydrolysate of γ-zein.The antihypertensive activities of synthetic Val-His-Leu-Pro-Pro and Leu-Pro-Pro were also investigated. These peptides, when intravenously administered to anesthetized rats at 125 mg/kg (Leu-Pro-Pro) or 160 mg/kg (Val-His-Leu-Pro-Pro), antagonized the rats’ pressor response to angiotensin I.

Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors.

Neutral endopeptidase (EC 3.4.24.11) from pig kidney hydrolyses [125I]iodo-insulin B-chain and leucine-enkephalin. Both activities were equally sensitive to inhibition by phosphoramidon [N-(alpha-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan] and thiorphan [N-(DL-2-benzyl-3-mercaptopropionyl)glycine]. Thermolysin hydrolysis of insulin B-chain was also sensitive to both inhibitors. The hydrolysis of the Gly3-Phe4 bond of Leu-enkephalin by synaptic membranes prepared from pig brain was partially inhibited by phosphoramidon and thiorphan. Synaptic membranes appear to contain another endopeptidase activity that is insensitive to these reagents. These observations suggest that enzymes similar to the kidney endopeptidase may play a general role in neuropeptide metabolism.

Primary structure of stellin B

Stellin B, a protamine fromAcipenser stellatus, has been subjected to thermolysin hydrolysis. Eight peptides have been isolated by chromatography on CM-Sephadex, and their structures have been determined by dansylation and by hydrolysis with carboxypeptidases A and B and with trypsin. From the results obtained, the complete amino acid sequence of stellin B has been established.

Conformational Analysis of Serum Apolipoprotein AII in Lipoprotein Complexes with Bifunctional Crosslinking Reagents

Apolipoprotein AII isolated from human serum high density lipoproteins was recombined with phosphatidylcholine to yield homogeneous particles of 80-120 A diameter. The radioactive bifunctional crosslinkers dimethyl [1,1'-14C]suberimidate and dimethyl 4,4'-dithiobis([1-14C]butyrimidate) were reacted with these particles. The kinetics of the reactions and the localisation of the crosslinked lysines of the polypeptide chains were determined. Thermolysin hydrolysis followed by two-dimensional separation of the peptides and isolation of the mono- and bifunctionally modified peptides allowed the assignment of the crosslinked peptides of the apoprotein AII seqeunce. The crosslinking pattern indicates a close-neighbour relationship (13-15 A) of the peptide chains between amino acid residues 3, 23, 46 and 55 of the symmetrical halves of the apo AII molecule. A reconstruction of the secondary structure of Apo AII in the lipoprotein complex on the basis of theoretical calculations is given and correlated with the chemical data.

Histone III: IV. AMINO ACID SEQUENCE OF HISTONE III OF THE TESTES OF THE CARP, LETIOBUS BUBALUS

Abstract Histone III of the testes of the carp, Letiobus bubalus, contains a serine residue in place of the cysteine found in calf thymus histone III. Examination of the cyanogen bromide peptides, as well as peptides derived by tryptic, chymotryptic, or thermolysin hydrolysis, revealed no other differences in amino acid composition. Thus histone III shows a highly conserved sequence from fish to calf, there being a difference of only 1 residue in the 135 of the protein. As in the case of calf thymus histone III, there are two specific sites of methylation of e-amino groups of lysine residues. Each site contains one, two or three methyl groups.