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Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Binding occurs through hydrogen bonding with Gln, Lys, His, Tyr and also several hydrophobic interactions.
Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC 50 values in the millimolar range.
Angiotensin converting enzyme ACE inhibitors are a new class of drugs which are critical in the treatment of hypertension and heart failure 1. Although the synthetic drugs are often prescribed due to their well-known role and convenience of access, they have undesirable effects, such as extreme low blood pressure, cough, bad tastes and allergic reactions.
Contrariwise, natural inhibitors regulate hypertension under mild conditions without significant side effects; hence, they can be considered as worthy alternatives to synthetic drugs 2.
ACE inhibitory peptides are the best known amongst various classes of regulatory bioactive peptides due to the development of functional foods with homeostasis role in the human body 3. ACE is a Zn-metallopeptidase that removes a dipeptide from the decapeptide angiotensin-I to produce the potent vasoconstriction octapeptide angiotensin-II 4.
Investigations to find more bioactive peptides, especially multifunctional ones have attracted the attention of researchers. The bioactive peptides are often inactive in their natural forms and when released by enzymatic hydrolysis, display their biological activity 3 , 5. So far, several ACE inhibitory peptides have been reported from hydrolysis of different food proteins, such as hen egg white lysozyme 4 , fish 6 , corn 7 , soybean 8 , milk 9 , casein 10 , etc.
These peptides are good candidates to regulate blood pressure and also to balance fluid and electrolytes in mammals. Zizyphus jujuba is a well-known medicinal plant with several bioactivities, such as antimicrobial 11 , antidiabetic 12 , 13 , hypoglycemic 13 , and analgesic Likewise, we recently reported some antioxidant peptides extracted from Z.
Although there are several reports on the presence of different bioactive compounds in this plant, there is no evidence indicating the presence of ACE inhibitory peptides. In the present study, from protein hydrolysate of Z. Investigating the inhibition pattern of these peptides showed that they inhibited the enzyme action by competitive mechanism.
As a complement to our experiment, homology modeling, molecular docking and nanoseconds ns molecular dynamics MD simulation were performed to provide an atomistic insight into the interaction of IER peptide due to the higher affinity with the ACE. All other chemicals used were of analytical grades. Proteins were extracted from Z. Briefly, jujuba fruits were soaked in water and then crushed in a porcelain mortar to attain a homogeneous semi-solid mixture. Samples separated by dialysis were lyophilized and their proteins were quantified by Bradford method 15 , using bovine serum albumin as the control.
Enzymatic hydrolysis is essential for releasing bioactive peptides. Accordingly, the extracted proteins were incubated with two commercial enzymes, papain and trypsin, and a combination of the two. Each enzyme was dissolved separately 0.
Elution was performed using 0. Activity of the peptide fractions for ACE inhibition was measured according to our previous study ACE inhibition percentage was obtained based on the following equation:. Experiments were performed in triplicates. The IC 50 values of the purified peptides were compared with that reported for captopril used as a positive control. After further purification, the most active peptides, F2 and F4, were selected to determine their amino acid sequences.
Desalting of the samples was carried out using ZipTips [Millipore]. The amino acid sequence was determined via de novo sequencing method. The inhibition patterns of the target peptides F2 and F4 were determined by Lineweaver-Burk plots.
Enzymatic assay was carried out using different concentrations 0. The inhibition patterns of the peptides were determined in the absence and presence of the inhibitory peptide at two concentrations of 0. The value of the inhibition constant K i was obtained from the negative x intercept of the secondary plot. The grid box dimensions were set to 60, 60 and 60 grid points centered on the zinc-binding site and a grid spacing of 0.
The protein-tripeptide complex was solvated in TIP3P water molecules inside a cubic box. After energy minimization, two separate position-restrained MD simulations were carried out to equilibrate the system.
Long range electrostatic interactions were computed using particle mesh by Ewald method Both short-range and Lennard-Jones interactions were calculated with a 1. The minimum energy conformation of the ACE-IER complex was extracted from the trajectory using free energy landscape FEL analysis and considered as a representative structure of the complex.
Proteins extracted from Z. Bioactive peptides were released by enzymatic digestion using trypsin, papain and a combination of the two. Primary assay showed that all hydrolysates had inhibitory effects on ACE activity. The percentages of ACE inhibitory activity were calculated to be According to the results, tryptic hydrolysate with the highest ACE inhibitory potential was used for peptide purification. Under the same condition, these peptides showed a higher ACE inhibitory activity in comparison with hydrolysate, which is related to the elimination of less effective fractions during peptide purification process.
The IC 50 value of captopril was calculated to be 2. Kinetic studies were performed in the absence and presence of 0. V max was found to be unchanged, but K m value was altered, which is an indication of competitive inhibitory pattern Fig. The K m resulted to be 0. Furthermore, at 0. Collectively, results imply that these peptides inhibited the enzyme by binding to the free enzyme, but not to the ACE-substrate complex. Since the inhibition pattern of the ACE inhibitory peptides is related to their inhibitory structures, we hypothesize that these peptides may have similar structures to the substrate; hence, they might compete with the substrate for binding to the same interaction site on the enzyme.
Most of the ACE inhibitory peptides show competitive inhibition patterns 31 , as is also seen in this study. By using secondary plots Fig. The inhibition constants Ki were obtained from the negative x intercepts of the secondary plots. Although the crystal structure of the rabbit ACE enzyme has not been determined so far, it shares a high degree of amino acid sequence identity Afterwards, five-hundred docking runs were performed using AutoDock 4.
As mentioned before, there is no crystal structure available for rabbit ACE. Calculating the probability of hydrogen bonds existence revealed more details on the residues involved in the IER binding to the enzyme active site Fig.
Throughout the simulation, four hydrogen bonds between IER and ACE within the docked structure were broken or significantly weakened, but a new network of hydrogen bonds was formed which stabilized the IER in its location within the ACE active site. The plots were generated using trial version of Mathematica 9 www. Red lines indicate the presence of a hydrogen bond at that specific time.
Each value of the hydrogen bond index corresponds to a unique donor-hydrogen-acceptor triplet. The amino acids involved in each particular hydrogen bond and the percentage of time that the hydrogen bond existed during the trajectory are given on the right side of the map. Two hydrogen bonds AlaIle1 and Arg3-Glu were broken and weaken, respectively. Structural comparison of the interaction networks of IER, captopril, enalaprilat and lisinopril with ACE active site indicates that the results of this study are in good agreement with those obtained for known competitive inhibitors of testicular ACE 34 , The other two inhibitors have a small methyl group instead of lysine.
Taken together, it seems that IER inhibits ACE in a competitive manner, as supported by experimental and structural evidences. The hydrophobic interactions are highlighted with green lines. The complex is also stabilized by the hydrophobic interactions with Glu, His, Phe and Tyr The equivalent residues in the human testicular ACE are enclosed in parentheses.
The phenyl moiety of these inhibitors which is absent in captopril mediates hydrophobic interactions with residues Ser and Val Analysing the inhibition mechanism by Lineweaver-Burk plots revealed that these peptides compete with the substrate in binding to the enzyme active site.
Salvetti, A. Angiotensin-converting enzyme inhibitors in hypertension: a review. Mane, S. Food Biochem. Li, G. Angiotensin I—converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Memarpoor-Yazdi, M. A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates. Foods 4 , — Antioxidant activity of protein hydrolysates and purified peptides from Zizyphus jujuba fruits.
Foods 5 , 62—70 Kohama, Y. Biological properties of angiotensin-converting enzyme inhibitor derived from tuna muscle.
Deutschland Deutsch English. Nucleic acid sample preparation involves a range of techniques to transform a sample which cannot be directly analyzed into one that fits the requirements of the analytical technique to be used such as reverse transcription RT or PCR. Nucleic acid sample purification and quality assessment are important steps in experimental workflows since the quality of nucleic acids can affect the performance in downstream reactions. Common nucleic acid purification techniques, as well as characterization methods, are discussed in the sections below. Related sections: Nucleic Acid Electrophoresis.
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In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus , due to its broad feedstock catabolism and high lipid yield.
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins e. Computational methods typically use computer programs to analyze proteins.
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Request PDF | On Mar 1, , Rodney Boyer published Principles and reactions of protein extraction, purification, and characterization.AnahГ C. 26.03.2021 at 07:56
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