INHIBITION LÀ GÌ

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Natural trypsin Inhibitors also known as serine protease inhibitors (serpins) are the largest và most diverse family of protease inhibitors.1 Serpins control the activation và catabolism of proteins by the inhibition of serine proteases in vivo.2

There are four natural sources of trypsin inhibitors: bovine pancreas, ovomucoid, soybean, và lima bean. Each inhibitor acts as a competitive substrate analog and binds with its serine protease to lớn size an inactive sầu complex, therefore rendering the protease inactive.3

This process allows the serpin (trypsin inhibitor) to lớn stop the proteolytic activity of the serine protease when its function is no longer necessary.

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Trypsin inhibitors provide unique processes depending on their source. For example, inhibitors in the seeds of legumes (soybean và lima bean) act as a feeding deterrent for insects by disrupting midgut proteases. This natural function is being expanded upon in the development of insect resistant transgenic plants. Soybean inhibitors have sầu also been found khổng lồ contribute lớn pancreatic hypertrophy in rats, again providing a feeding deterrent. The Bowman-Birk soybean inhibitor is being studied as a cancer preventive agent.4

Products


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Figure 1.T0256 - Trypsin inhibitor from bovine pancreas


Synonym: BPTI (basic pancreatic trypsin inhibitor)M.W.: ~6.5 kDa (single-chain 58-amino acid peptide)pI: ~10.5Specific Activity: One mg of trypsin inhibitor will inhibit greater than 1.5 mg of trypsin with activity of ~10,000 BAEE units per mg proteinDerived from New Zealand-sourced pancreasSolubility: This product is soluble in water (2 mg/mL)

BPTI is a 58 amino acid single polypeptide chain with 3 disulfide bonds.5

BPTI inhibits both bovine & human trypsin, chymotrypsin, kallikrein và plasmin. BPTI does not inhibit porcine elastase.6


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Figure 2. T9253 - Trypsin inhibitor from chicken egg White (Type II-O)


Synonym: OvomucoidM.W.: ~28 kDapI: 4.17Specific Activity: One mg will inhibit 0.8-1.2 mg of trypsin with activity of ~10,000 BAEE units per mg protein. May inhibit ≤ 0.3 mg of chymotrypsin with activity of approx. 40 BTEE units per mg protein.Solubility: This product is soluble in 0.67 M phosphate buffer, pH 7.6 (2 mg/mL).

Chicken ovomucoid is a major glycoprotein that inhibits bovine trypsin. It is comprised of 186 amino acids that are arranged in three tandem domains.8 Each domain contains three disulfide bonds, two tyrosine residues, và one active sầu site.9 Ovomucoid is described as a single headed inhibitor of trypsin, meaning each ovomucoid molecule binds 1:1 with trypsin.7

This sản phẩm contains some ovoinhibitor. Ovoinhibitor is another protease inhibitor from chicken egg white. Ovoinhibitor has at least five binding sites, và is responsible for the inhibition of bovine trypsin, bovine chymotrypsin & porcine elastase.10 Trypsin & chymotrypsin comprise 2 binding sites each, while the last site binds elastase.11 Ovomucoid và Ovoinhibitor are the two most abundant protease inhibitors in chicken egg trắng, comprising 11 và 1.5% of the protein.12


T2011 - Trypsin inhibitor from chicken egg white

Type III-O (free of ovoinhibitor)Synonym: OvomucoidM.W.: ~27 kDaSpecific Activity: One milligram of product will inhibit 1.0-2.0 mg of trypsin with activity of ~10,000 BAEE units/mg proteinSolubility: This sản phẩm is soluble in 0.67 M phosphate buffer, pH 7.6 (2 mg/mL)

This sản phẩm has the same characteristics as described above for product T9253, but is further purified by an ammonium sulfate cut and filtration process to eliminate the ovoinhibitor. The absence of ovoinhibitor results in an even more pure ovomucoid that continues lớn inhibit trypsin in a 1:1 complex.

T4385 - Trypsin inhibitor from turkey egg white

Type II-TSynonym: Turkey OvomucoidM.W.: ~20 kDaSpecific Activity: One mg will inhibit 0.9-1.3 mg of trypsin with activity of ~10,000 BAEE units per mg protein or 0.4-1.0 mg of α-chymotrypsin with activity of ~40 BTEE units per mg proteinSolubility: This product is soluble in 0.67 M phosphate buffer, pH 7.6 (1 mg/mL)

Trypsin inhibitor from turkey egg white contains two independent binding sites, one for bovine trypsin & the other for α-chymotrypsin. At low ph (2.0) a third domain (OMTKYT3) develops & inhibits most serine proteases that prefer a neutral complex site.13


Kunitz & Bowman-Birk (BBI) Soybean Protease Inhibitors

These two proteins are the most abundant protease inhibitors in soybeans. BBI with a molecular mass of 8 kDa is a strong inhibitor of both trypsin and chymotrypsin và contains independent binding sites for each. The Kunitz inhibitor with a molecular mass of 20.1 kDa, comprises one binding site that strongly inhibits trypsin while weakly binding chymotrypsin.


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Figure 3.Trypsin inhibitor from Glycine max (soybean): Kunitz Inhibitor


Synonyms: Kunitz Trypsin Inhibitor, Tia1, STI và SBTIM.W.: đôi mươi.1 kDapI: 4.525Extinction Coefficient: E1% = 9.94(280 nm, pH 7.6 buffer)

Soybean trypsin inhibitor was first isolated by Kunitz.14 Several other related inhibitors are also found in soybeans.15 Trypsin inhibitor from soybeans is a monomeric protein containing 181 amino acid residues in a single polypeptide chain cross linked by two disulfide bridges.16-18

Soybean trypsin inhibitor inhibits trypsin, & to a lesser extent chymotrypsin19 and plasmin.20 Soybean trypsin inhibitor will also inhibit other proteases by a mechanism similar to trypsin. SBTI has inhibitory effects towards plasma kallikrein & coagulation Factor Xa. However, Soybean trypsin inhibitor will not inhibit metalloproteases, tissue-based kallikrein, acid proteases, or thio proteases.

Soybean trypsin inhibitor forms a 1:1 stoichiometric complex with the protease active site. Upon formation of this complex, trypsin may cleave sầu a single arginine-isoleucine bond on the inhibitor.21,22 Inhibition is both reversible & pH dependent. Dissociation of this complex may yield a modified or native sầu size of the inhibitor.23 The optimal pH for trypsin binding is 8.0 with an association constant of greater than 109 at pH 8.0 and an association constant 0.15-2.6 x 104 at pH 3.6-4.4.24


T9003 - Trypsin inhibitor from Glycine max (soybean)Type I-SThis product is chromatographically purified on DEAE Sepharose & contains 10% Sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg will inhibit 1.0-3.0 mg of trypsin with activity of ~10,000 BAEE units per mg protein.One chymotrypsin unit will hydrolyze 1.0 µmole of BTEE per min at pH 7.8 at 25 °C.Solubility: Trypsin inhibitor is soluble in water & phosphate buffers at 10 mg/mL. It is soluble in balanced salt solutions (1 mg/ml) và in serum-free media. Solutions at concentrations higher than 10 mg/ml may be hazy và have a yellow to lớn amber color.Storage/Stability: This product remains active in frozen aliquots at –đôi mươi °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating khổng lồ 80 °C and irreversibly denatured by heating to lớn 90 °C.15

T6522 - Trypsin inhibitor from Glycine max (soybean)Type I-SCell Culture testedThis sản phẩm is chromatographically purified on DEAE Sepharose and contains 10% Sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg will inhibit 1-3 mg of trypsin with activity of ~10,000 BAEE units per mg proteinSolubility: Trypsin inhibitor is soluble in water and phosphate buffers at 10 mg/mL. It is soluble in balanced salternative text solutions (1 mg/mL) và in serum-không tính phí media. Solutions at concentrations higher than 10 mg/mL may be hazy & have a yellow to amber color.Storage/Stability: This product remains active in frozen aliquots at –đôi mươi °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating khổng lồ 80 °C and irreversibly denatured by heating lớn 90 °C.15

T6414 - Trypsin inhibitor from Glycine max (soybean)This hàng hóa is a sterile filtered, cell cultured 1x solution (0.1% trypsin inhibitor solution in Dulbecco"s PBS), which is appropriate for use in cell culture applications. It has been optimized for passage of endothelial cells.Storage/Stability: This sản phẩm remains active sầu in frozen aliquots at –trăng tròn °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to lớn 80 °C and irreversibly denatured by heating khổng lồ 90 °C.15

T9008 - Trypsin inhibitor from Glycine max (soybean) 1% solution in waterFor use in cell culture applicationsPrepared from T9003, sterile filtered và processed lớn yield a convenient no buffer solution.Storage/Stability: This product is stored at 2-8 °C. This solution can also be stored as frozen aliquots at –đôi mươi °C, but freeze-thaw cycles should be avoided. This protein is reversibly denatured by short heating to lớn 80 °C & irreversibly denatured by heating khổng lồ 90 °C.15


T9128 - Trypsin inhibitor from Glycine max (soybean) Type II-SPrepared by ammonium sulfate fractionations along with a series of clarification steps to lớn yield a sản phẩm with 90% protein & 10% Sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg will inhibit a minimum of 1.0 mg of trypsin with activity of ~10,000 BAEE units per mg protein.Solubility: This sản phẩm is soluble in water (1 mg/mL). Solutions at higher concentrations (greater than 10 mg/mL) may be hazy & have sầu a yellow to amber color.Storage/Stability: This hàng hóa remains active in frozen aliquots at —20 °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to lớn 80 °C & irreversibly denatured by heating lớn 90 °C.15

T2327 - Trypsin inhibitor from Glycine Max (soybean)≥98% Kunitz type inhibitorFurther purified chromatographically from T9128 to lớn yield pure Kunitz Type trypsin inhibitor.This hàng hóa contains 10% sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg will inhibit ≥ 1.6 mg of trypsin with activity of ~10,000 BAEE units per mg protein.Solubility: Trypsin inhibitor is soluble in water & phosphate buffers at concentrations higher than 10 mg/mL may be hazy và have sầu a yellow to lớn amber color.Storage/Stability: This hàng hóa remains active sầu in frozen aliquots at -20 °C, but freeze-thaw should be avoided. This protein is reversibly denatured by short heating to lớn 80 °C & irreversibly denatured by heating khổng lồ 90 °C.15

Trypsin-chymotrypsin inhibitor from Glycine max (soybean): Bowman-Birk inhibitor


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Figure 4.T9777 - Trypsin-chymotrypsin inhibitor from Glycine max (soybean)


Synonym: Bowman–Birk inhibitor (BBI)CAS Number: 37330-34-0Unit definition: One trypsin unit will produce a ΔA253 of 0.001 per min with BAEE as substrate at pH 7.6 at 25 °C; reaction volume 3.2 ml, 1 cm light path.M.W.: 8 kDa29

The Bowman-Birk inhibitor (BBI) from soybean is a monomeric protein containing 71 amino acids in a single polypeptide chain crosslinked by seven disulfide bridges.26 This inhibitor contains two independent inhibitory binding sites, one for trypsin và the other for chymotrypsin.27 BBI binds each protease to khung a 1:1 complex. Since the inhibition is non-competitive sầu, BBI has the ability to lớn form a ternary complex with both enzymes.28

This product is chromatographically purified on DEAE Sepharose và contains 20% Sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg protein will inhibit ≥0.5 mg trypsin with activity of ~10,000 BAEE units per mg protein.One mg protein will inhibit ≥1.0 mg chymotrypsin with activity of ~40 BTEE units per mg protein.Solubility: Trypsin-chymotrypsin inhibitor is soluble in water or 0.67 M Sodium phosphate, pH 7.6 (1 mg/mL).

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Trypsin inhibitor from Phaseolus limensis (lima bean):

Synonyms: Trypsin Inhibitor from lima beans và LBTIM.W.: 9 kDapI: 4.525Extinction Coefficient: E1% = 9.94(280nm, pH 7.6 buffer)

Trypsin inhibitor from lima bean is a monomer comprised of 83 amino acids that has the ability lớn undergo a concentration-dependent dimerization. The degree of self-association depends upon the type of variant & pH.30

Trypsin inhibitor from Phaseolus limensis has four khổng lồ six variants whose inhibitory activities towards trypsin are essentially identical; whereas, some differences exist towards inhibition of chymotrypsin. The variant’s amino acid sequences are similar, each containing 7 disulfide bonds.31

Trypsin inhibitor forms a 1:1 stoichiometric complex with the protease active sầu site. The complex of the inhibitor with either trypsin or chymotrypsin has no further inhibitory effect toward more of the same enzyme, but has full activity towards the other enzyme (forming 1:1:1 complex).30,32 This implies that there is one binding site for trypsin và another for chymotrypsin. Trypsin binds to lớn a Lys-Ser site, while chymotrypsin binds lớn a Leu-Ser site.31 Inhibition is both reversible & pH dependent. Dissociation of this complex may yield a modified or native sầu size of the inhibitor.23 The optimal pH for trypsin binding is 8.0 with an association constant of greater than 109 at pH 8.0, và an association constant of 0.15-2.6 x 104 at pH 3.6-4.4.24


T9378 Trypsin inhibitor from Phaseolus limensis (lima bean)This hàng hóa is a lyophilized powder containing 10% Sodium phosphate buffer salts, pH 7.6.Specific Activity: One mg will inhibit a minimum of 0.8 mg of trypsin with activity of ~10,000 BAEE units per mg protein.Solubility: Trypsin inhibitor is soluble in water và phosphate buffers at 1 mg/mL. It is soluble in balanced salternative text solutions & in serum-không lấy phí media. Solutions at concentrations higher than 10 mg/mL may be hazy and have a yellow to amber color.


Cell Culture Application

Trypsin inhibitors are used in cell culture applications khổng lồ further inhibit tryptic activity during cell dissociation lớn prsự kiện cell damage/death.

Procedure:After trypsinizing cells, resuspkết thúc cells in 1 mL trypsin inhibitor solution (1 mg/mL using either a balanced salt solution or serum free media) for every mL of trypsin solution used for dissociation. Centrifuge the cell suspension at 1000 rpm for 5 minutes. A cell pellet should khung. Remove sầu as much of the trypsin inhibitor as possible & resuspend the pellet in serum-không tính phí medium. Culture cells as desired.


Assay Method for Trypsin Inhibitor Activity

The activity of most trypsin inhibitor preparations is determined by a continuous rate spectrophotometric assay & expressed as the inhibition of BAEE units.

Unit Definition: One BAEE unit will produce a ΔA253 of 0.001 per min at pH 7.6 & 25 °C using BAEE as a substrate. Reaction volume = 3.2 mL.

ConditionsTemp = 25 °C, pH = 7.6, A= 253 nm, Light path = 1 cmIn a 3.2 mL reaction phối, the final concentrations are 63 mM sodium phosphate, 0.23 mM Nα-benzoyl-L-arginine ethyl ester (BAEE), 0.002 mM hydrochloric acid, 0.005mg trypsin, and 0.003 - 0.001 mg trypsin inhibitor.

Reagents NeededS0751 - Sodium Phosphate monobasicB4500 - Nα-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE)258148 - Hydrochloric acid ACS reagentT8003 - Trypsin from bovine pancreas

Reagents

67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C(Prepare 100 mL in deionized water using Sodium Phosphate, Monobasic, Anhydrous, Product No. S0751. Adjust khổng lồ pH 7.6 at 25 °C with 1 M NaOH.)0.25 mM Nα-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE)(Prepare 50 mL in Reagent a using Nα-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Product No. B4500.)1 mM Hydrochloric Acid Solution (HCl)(Prepare 50 mL in deionized water using concentrated Hydrochloric Acid, Product No. 258148.)Trypsin Enzyme Solution (Trypsin)(Immediately before use, prepare a solution containing 1 mg protein/mL of Trypsin, Product No. T8003, in cold Reagent C.)Trypsin Inhibitor Solution (Inhib.)(Immediately before use, prepare a solution containing 1.0 mg/mL of Trypsin Inhibitor in cold Reagent A.)

ProcedurePipette (in milliliters) the following reagents inlớn suitable quartz cuvettes

Part A:


Allow to lớn stand at 25 °C for a minimum of five minutes và no longer than six minutes.

Mix by inversion và pipette (in milliliters) the following reagents inkhổng lồ suitable cuvettes:

Part B:


Mix by inversion & equilibrate khổng lồ 25 °C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:


Immediately mix by inversion và record the increase in A253nm for approximately 5 minutes. Obtain the ΔA253nm/minute using the maximum linear rate for the Tests, Blank, and Uninhibited Solution.Calculation


Trypsin Activity in BAEE units/mL enzyme =

(ΔA253nm/min Test – ΔA253nm/min Blank)(df)(10.0)

(0.001)(0.10)(0.5)


df = Dilution factor 0.001 = The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25 °C in a 3.2 ml reaction mix 0.10 = Volume (in milliliters) enzyme used (Part B) 10.0 = Total volume in milliliters of assay (Part A) 0.5 = Volume (in milliliters) of enzyme used (Part A)


Plot the Trypsin activity (BAEE units/mg protein) vs mL of Trypsin Inhibitor/RMMg Trypsin Inhibitor = (mL of Trypsin Inhibitor)(Conc. of Trypsin Inhibitor, mg/mL)


Mg Trypsin Inhibited by 1 mg Trypsin Inhibitor =mg Trypsin/RM (normalizing factor)mg Trypsin Inhibitor (from plot)

Normalizing Factor = (BAEE Units of Uninhibited Trypsin per mg solid/10,000 BAEE units of Trypsin per specification.)

Notes:

This enzyme assay is used lớn assay hàng hóa numbers: T9003, T9008, T9128, T9253, T2011, T4385, T9378, và T0256.When assaying Trypsin Inhibitor, Type II-S, hàng hóa number T9128, prepare a solution containing 0.60 mg/ml of Trypsin Inhibitor in cold Reagent a.The uninhibited Trypsin activity should be within 85% of the release value for activity.With 11,700 khổng lồ 13,005 Trypsin units/mg solid per label, the acceptable range for activity of the uninhibited Trypsin reaction should be 10,000 lớn 15,300 Trypsin units/mg solid. This range should also correspond lớn a corrected ΔAbs253nm/minute of 0.0545 khổng lồ 0.0835. With this rate & an inhibition of 20% lớn 80% the ΔAbs253nm/minute should be above the spectrophotometer rate detection limit of 0.00đôi mươi.

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Trypsin Unit Conversions 1 BAEE µM Unit = 200 BAEE Units1 TAME µM Unit = 0.27 BAEE µM Units1 BAEE µM Unit = 3.64 TAME Units1 TAME µM Unit = 55 BAEE A253 Units1 BAEE A253 Unit = 0.018 TAME µM Unit1 TAME µM Unit = 180 TAME A247 Units1 TAME A247 Unit = 0.33 BAEE Units1 USP Unit = 3.0 BAEE Units1 NF Unit = 1.1 USP Units


RAWLINGS ND, TOLLE DP.., BARRETT AJ. 2004. Evolutionary families of peptidase inhibitors. 378(3):705-716. http://dx.doi.org/10.1042/bj20031825
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PGW, Irving JA, Lomas DA, Luke CJ, Moyer RW, et al. 2001. The Serpins Are an Expanding Superfamily of Structurally Similar but Functionally Diverse Proteins. J. Biol. Chem.. 276(36):33293-33296. http://dx.doi.org/10.1074/jbc.r100016200
Zhou J, Liu C, Tsou C. 1989. Kinetics of trypsin inhibition by its specific inhibitors. Biochemistry. 28(3):1070-1076. http://dx.doi.org/10.1021/bi00429a022
Kennedy AR. 1998. The Bowman-Birk inhibitor from soybeans as an anticarcinogenic agent. 68(6):1406S-1412S. http://dx.doi.org/10.1093/ajcn/68.6.1406s
Huber R, Kukla D, Ruhlmann A, Steigemann W. 1972. Pancreatic Trypsin Inhibitor (Kunitz): Part I: Structure and function. Cold Spring Harbor Symposia on Quantitative Biology. 36(0):141-150. http://dx.doi.org/10.1101/sqb.1972.036.01.019
Pancreatic trypsin inhibitor precursor - Bos taurus (Bovine). . Universal Protein Resource (UniProt).. Available from: https://www.uniprot.org/uniprot/P00974