scholarly journals Dipeptidyl Peptidase-IV Inhibitory Activity and Related Molecular Mechanism of Bovine α-Lactalbumin-Derived Peptides

Molecules ◽  
2020 ◽  
Vol 25 (13) ◽  
pp. 3009
Author(s):  
Jing Gao ◽  
Han Gong ◽  
Xueying Mao
Keyword(s):  
Liquid Chromatography ◽  
Inhibitory Activity ◽  
High Performance ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Salt Bridges ◽  
Docking Analysis ◽  
Inhibitory Peptides ◽  
Dpp Iv

Identifying DPP-IV inhibitory peptides from dietary protein has attracted increased attention. In the present study, bovine α-lactalbumin hydrolysates were generated by alcalase for various hydrolysis times, and DPP-IV inhibitory activity of these hydrolysates was determined. The 4 h hydrolysates displayed the most potent DPP-IV inhibitory activity, with DPP-IV inhibition rate of 82.30 ± 1.39% at concentration of 1.0 mg/mL. DPP-IV inhibitory peptides were isolated from the 4 h-hydrolysates with gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). Using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS), two DPP-IV inhibitory peptides were identified, and their amino acid sequences were Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr (ELKDLKGY) and Ile-Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr (ILDKVGINY), respectively. Furthermore, molecular docking analysis showed that peptides ELKDLKGY and ILDKVGINY could form hydrogen bonds, pi-cation interactions, and salt bridges with DPP-IV. These findings indicated that bovine α-lactalbumin may be a potential source of natural DPP-IV inhibitor.

The use of reversed-phase high-performance liquid chromatography to detect proteolytic activity from human pancreas in a radioimmunoassay for corticotrophin-releasing factor

1987 ◽  
Vol 114 (1) ◽  
pp. 147-151 ◽  
Author(s):  
D. S. Jessop ◽  
R. L. Patience ◽  
D. Cunnah ◽  
L. H. Rees
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Proteolytic Activity ◽  
High Performance ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Pancreatic Tissue ◽  
Acid Extraction ◽  
Human Pancreas ◽  
Corticotrophin Releasing Factor

ABSTRACT Degradation of tracer during a radioimmunoassay (RIA) can result in false-positive concentrations of immunoreactivity being reported in a biological sample. A technique has been developed using reversed-phase high-performance liquid chromatography (HPLC) to detect proteolytic degradation of corticotrophin-releasing factor-41 (CRF-41) during incubation with tissue extracts under RIA conditions. Human pancreatic tissue was extracted in HCl or urea and incubated with 125I-labelled CRF-41 at neutral pH for 18 h. When samples were analysed by HPLC and fractions counted for radioactivity, tracer was extensively degraded. Heating extracts at 85 °C or adding lima bean trypsin inhibitor to the medium prevented degradation. Pancreatic tissue extracted in HCl was analysed by gel filtration and HPLC, and fractions were subjected to RIA for CRF-41. A peak of immunoreactivity was detected by both chromatographic methods. However, when this material was incubated with tracer and analysed by HPLC, the tracer was degraded, indicating that proteolytic activity remained after acid extraction and two forms of chromatography. J. Endocr. (1987) 114, 147–151

scholarly journals Purification and Identification of Novel Xanthine Oxidase Inhibitory Peptides Derived from Round Scad (Decapterus maruadsi) Protein Hydrolysates

Marine Drugs ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. 538
Author(s):  
Xiao Hu ◽  
Ya Zhou ◽  
Shaobo Zhou ◽  
Shengjun Chen ◽  
Yanyan Wu ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Inhibitory Activity ◽  
High Performance ◽  
Amino Acid Sequences ◽  
Inhibitory Effects ◽  
Small Peptides ◽  
Inhibitory Peptides ◽  
Metal Affinity ◽  
Fluorescence Quenching Mechanism ◽  
Decapterus Maruadsi

The objective of the present study was to investigate the xanthine oxidase (XO) inhibitory effects of peptides purified and identified from round scad (Decapterus maruadsi) hydrolysates (RSHs). In this study, RSHs were obtained by using three proteases (neutrase, protamex and alcalase). Among them, the RSHs of 6-h hydrolysis by neutrase displayed the strongest XO inhibitory activity and had an abundance of small peptides (<500 Da). Four novel peptides were purified by immobilized metal affinity chromatography and identified by nano-high-performance liquid chromatography mass/mass spectrometry. Their amino acid sequences were KGFP (447.53 Da), FPSV (448.51 Da), FPFP (506.59 Da) and WPDGR (629.66 Da), respectively. Then the peptides were synthesized to evaluate their XO inhibitory activity. The results indicated that the peptides of both FPSV (5 mM) and FPFP (5 mM) exhibited higher XO inhibitory activity (22.61 ± 1.81% and 20.09 ± 2.41% respectively). Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by these inhibitors (FPSV and FPFP) was a static quenching procedure. The study of inhibition kinetics suggested that the inhibition of both FPSV and FPFP was reversible, and the type of their inhibition was a mixed one. Molecular docking revealed the importance of π-π stacking between Phe residue (contained in peptides) and Phe914 (contained in the XO) in the XO inhibitory activity of the peptides.

Angiotensin I–Converting Enzyme Inhibitory Activity of Peptides Derived from Egg White Proteins by Enzymatic Hydrolysis

2004 ◽  
Vol 67 (9) ◽  
pp. 1914-1920 ◽  
Author(s):  
M. MIGUEL ◽  
I. RECIO ◽  
J. A. GÓMEZ-RUIZ ◽  
M. RAMOS ◽  
R. LÓPEZ-FANDIÑO
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Molecular Mass ◽  
Inhibitory Activity ◽  
High Performance ◽  
Egg White ◽  
Reversed Phase ◽  
Converting Enzyme ◽  
Ace Inhibitory Activity ◽  
Ace Inhibitory

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 μg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 μg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 &lt; 40 μg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography–tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 μM, respectively.

scholarly journals Kappacin, a Novel Antibacterial Peptide from Bovine Milk

2001 ◽  
Vol 45 (8) ◽  
pp. 2309-2315 ◽  
Author(s):  
Marina Malkoski ◽  
Stuart G. Dashper ◽  
Neil M. O'Brien-Simpson ◽  
Gert H. Talbo ◽  
Mary Macris ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
High Performance ◽  
Active Form ◽  
Bovine Milk ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Inhibitory Peptide ◽  
Rp Hplc ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity

ABSTRACT Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans andPorphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated κ-casein (residues 106 to 169) [κ-casein(106–169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)149κ-casein-A(138–158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)149κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)149 κ-casein-A(138–158) displayed growth-inhibitory activity against S. mutans(MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Purification and Biochemical Characterization of Trypsin Inhibitor from Oyster

2012 ◽  
Vol 577 ◽  
pp. 119-124
Author(s):  
Yuan Hui Zhao ◽  
Ming Yong Zeng ◽  
Xia Li
Keyword(s):  
Liquid Chromatography ◽  
Trypsin Inhibitor ◽  
High Performance ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Phase Liquid ◽  
Lung Adenocarcinoma A549 ◽  
Head Inhibitor

In this paper, the purification and biochemical characterization of the endogenous oyster (Crassostrea gigas) trypsin inhibitor were researched. A oyster trypsin inhibitor（OTI）has been purified by successive ammonium sulfate precipitation, gel filtration, affinity chromatography and high performance reversed-phase liquid chromatography. OTI has a molecular weight of approximately 5036 Da estimated by high performance size exclusive liquid chromatography. OTI was heat-, acid- and basic-stable competitive trypsin inhibitor. And OTI was double-head inhibitor with the inhibition constant (Ki) value of 1.644×10-2 mmol L-1. OTI was composed of nine kinds of amino acid, and rich in cysteine, alanine and glutamic acid. Furthermore, OTI can inhibit the proliferations of human lung adenocarcinoma A549 cell and human cervical cancer Hela cell

CHARACTERISATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF CIRCULATING GROWTH HORMONE RELEASING FACTORS IN A HUMAN PLASMA

1985 ◽  
Vol 105 (1) ◽  
pp. R1-R4 ◽  
Author(s):  
E.S. Penny ◽  
R.L. Patience ◽  
A.M. Sopwith ◽  
J.A.H. Wass ◽  
G.M. Besser ◽  
...  
Keyword(s):  
Growth Hormone ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Carcinoid Tumour ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Molecular Forms ◽  
Elevated Concentration

ABSTRACT Three forms of circulating immunoreactive human growth hormone-releasing factor (ir-hGRF) have been identified from a patient whose acromegaly was associated with a disseminated carcinoid tumour. This is the first known report of the molecular forms of ir-hGRF in human plasma. High performance liquid chromatography (HPLC) on a C3, wide pore reversed-phase column and gel filtration chromatography were used in conjunction with a sensitive radioimmunoassay (RIA). The greatly elevated concentration of the ir-hGRF in plasma from this patient was 25,000 ng/l (normal range <60 ng/l). Gel filtration (G50) chromatography of the plasma revealed a single peak which coeluted with synthetic hGRF-40. However, reversed-phase HPLC of Vycorextracted plasma resolved the ir-hGRF into three components, which coeluted with synthetic hGRF-40 (69%), hGRF-44 (22%) and hGRF-37 (9%). At present it is not clear if the three forms are natural variants or whether either or both hGRF-40 and hGRF-37 are cleavage products of hGRF-44.

scholarly journals Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius)

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 3
Author(s):  
Laura C. Wait ◽  
Andrew A. Walker ◽  
Glenn F. King
Keyword(s):  
Liquid Chromatography ◽  
High Performance ◽  
Sulfhydryl Group ◽  
Venom Gland ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Mass Spectrometry Data ◽  
Cub Domain ◽  
Liquid Chromatography Tandem Mass ◽  
Assassin Bug

Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (Havinthus rufovarius), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (Lucilia cuprina). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis, their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

scholarly journals Screening of an Endophyte Transforming Polydatin to Resveratrol from Reynoutria Japonica Houtt and the Optimization of Its Transformation Parameters

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4830
Author(s):  
Jin Liu ◽  
Xueqing Zhang ◽  
Ting Yan ◽  
Faling Wang ◽  
Jing Li ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Inhibitory Activity ◽  
High Performance ◽  
Ethyl Acetate Fraction ◽  
Reversed Phase ◽  
Transformation Rate ◽  
Inhibition Activity ◽  
Phylogenetic Tree Analysis ◽  
Glucose Content ◽  
Reynoutria Japonica

Resveratrol showed various kinds of bioactivities, such as antioxidant, antimicrobial, anticancer effects and, therefore, has been used widely as an important ingredient in medication, healthy foods and cosmetics. However, in nature, resveratrol usually exists at low content and more often exists as polydatin. Therefore, it becomes important to find the cost-effective and environmental-friendly way to transform polydatin to resveratrol. In this study, endophytes were isolated from the rhizome tissue of Reynoutria japonica and screened for transforming polydatin to resveratrol using reversed-phase high-performance liquid chromatography (RP-HPLC) and confirmed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. A bacterium identified as Bacillus aryabhattai using 16S rRNA phylogenetic tree analysis showed highest transformation rate. The transforming conditions were optimized including substrate concentration, substrate addition time, culture temperature and inoculation ratio. Our results demonstrated that the bacteria isolated from R. japonica rhizome tissue showed high activity in transforming polydatin into resveratrol. Crude extract of R. japonica root and rhizome (RJE) was also tested as substrate and it was found that the transformation was significantly inhibited at 10.0 mg/mL RJE. Emodin at equivalent concentration of 10.0 mg/mL RJE showed no inhibition activity, and glucose content in RJE was trace and far from enough to exhibit the inhibitory activity. Successive solvent partition followed by an inhibition activity assay revealed that the ethyl acetate fraction showed the main inhibition activity. However, due to the coexistence of polydatin and compounds with inhibitory activity, the concentration of RJE can only be used at limited concentration as substrate.

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