抑肽酶Aprotinin from Bovine Lung

• 产品编号：0219410105      品牌：MP       原厂货号：0219410105
• 产品分类：蛋白质检测与分析 > 蛋白质，多肽和酶 > 蛋白酶
• 应用分类：

描述：

(EC 3.4.21.9)
From Bovine Lung
Protease Inhibitor
Activity: ≥3 TIU/mg.
Unit Definition: One trypsin inhibitor unit (TIU), which is equal to 900 kallikrein inhibitor units (KIU) and 8,800 Schwert and Takenake units (STU), is the amount of inhibitor per mg which hydrolyzes one μmole of benzoyl-DL-arginine-p-nitroanilide (BAPNA) per minute. One TIU reduces the activity of two trypsin units by 50%.
It is reactive against trypsin, chymotrypsin, plasmatic and glandular kininogenases, plasmin, urokinase, clotting factor XIIa, protein C, proteinases of the complement system, and leukocyte and tissue proteinases.

Description: Found in bovine lymph nodes, lung, parotid gland, spleen, liver, pancreas, seminal vesicles, thyroid gland, kidney, mucous membranes of the trachea and esophagus, ovaries, heart, posterior pituitary and cartilage.^9,17,18

A serine protease inhibitor which is reactive against trypsin, chymotrypsin, plasmatic and glandular kininogenases, plasmin, kallkrein, urokinase, clotting factor XIIa, protein C, proteinases of the complement system, and leukocyte and tissue proteinases. It does not inhibit thrombin. Aprotinin works by blocking the active sites of enzymes. Binding is reversible with most aprotinin-protease complexes dissociating at pH > 10 or < 3.

Effective Concentration: Equimolar with protease (1-2 ug/ml).

Stability: Aprotinin is relatively stable to high temperature, acids, alkali, organic solvents and proteolytic digestion (only thermolysin has been found capable of degrading aprotinin after heating to 60-80°C).¹⁸ The Cyc¹⁴-Cys³⁸ disulfide bridge is readily split by reducing agents like b-mercaptoethanol.¹⁸

TIU Assay Procedure:

Principle:

BAPNA = Na-Benzoyl-DL-Arginine-p-nitroanilide

Conditions: T = 25°C, pH = 7.8, A405nm, Light path = 1 cm

Method: Continuous spectrophotometric determination.

Reagents:
 

A. 200 mM Triethanolamine buffer with 20 mM calcium chloride, pH 7.8 at 25°C: Prepare 100 ml in deionized water using triethanolamine hydrochloride and calcium chloride, dihydrate. Adjust to pH 7.8 at 25°C with 1 M NaOH.
B. 0.1% (w/v) Na-Benzoyl-DL-arginine-p-arginine-p-nitroanilide solution (BAPNA): Prepare 25 ml in deionized water using Na-Benzoyl-DL-arginine-p-nitroanilide, hydrochloride. Heat solution to not greater than 65°C to facilitate solubilization. Prepare solutions fresh each time. Solutions must be completely solubilized before use. Note: If the solution is hazy, continue to stir over gentle heat until the solution becomes clear. Do not use the solution if it turns yellow - this indicates possilbe chemical decomposition of the substrate due to overheating.
C. 1 mM Hydrochloric acid solution: Prepare 50 ml in deionized water.
D. Trypsin Enzyme Solution: Prepare by dissolving 2.5 mg Trypsin in 20 ml of cold reagent C. Prepare fresh each time.
E. 0.9% (w/v) sodium chloride solution (NaCl): Prepare 100 ml in deionized water.
F. Aprotinin Inhibitor Solution: Prepare three separate aprotinin solutions in Reagent E, each containing 0.047 to 0.06 trypsin inhibitor units per ml. Use a separate solution for each replication of assay. Note: The % inhibition must be between 40 and 60 percent for the assay to be valid. Adjust the concentration of the inhibitor solution so that the results are obtained in this range.

Procedure:

Pipette the following reagents into suitable cuvettes:

	Uninhibited Test	Inhibited Test	Blank
Reagent A	1.60 ml	1.60 ml	1.60 ml
Reagent C	--	--	0.20 ml
Reagent D	0.20 ml	0.20 ml	--
Reagent E	0.20 ml	--	0.20 ml
Reagent F	--	0.20 ml	--

Mix by inversion and equilibrate to 25°C. Monitor the A_405nm until constant, using a suitably thermostatted spectrophotometer. Then add:

 

Immediately mix by inversion and record the increase in A_405nm for approximately 5 minutes. Obtain the DA_405nm/minute using the maximum linear rate for the Uninhibited, Inhibited, and Blank Solutions.

Calculations:


 

TIU = Trypsin Inhibitor Units
df = Dilution factor
9.96 = The millimolar extinction coefficient of p-Nitroaniline at 405 nm
RM = Reaction Mix

Note: In cases where there is variability in the results, ensure that the uninhibited rate has a DA of 0.08 to 0.12. This may be required to reduce the variance caused by the range in specific activity of the Trypsin used.

Final Assay Concentration:

In a 3.00 ml reaction mix, the final concentrations are 107 mM triethanolamine, 11 mM calcium chloride, 0.03% (w/v) BAPNA, 0.07 mM hydrochloric acid, 0.025 mg trypsin, 0.12% (w/v) sodium chloride, 0.0003% (w/v) thimerosal and 0.0094-0.012 trypsin inhibitor unit of aprotinin.

 

原厂资料：

注意事项：

Unit Definitions:
 

KIU: One KIU (Kallikrein Inactivating Unit) is identical to the quantity of protease inhibitor that has the ability to inhibit 2 Kallikrein units by 50% under optimal conditions.

TIU: One trypsin inhibitor unit (TIU) is the amount of inhibitor per mg which hydrolyzes one umole of benozyl-DL-arginine-p-nitroanilide (BAPNA) per minute. One TIU reduces the activity of two trypsin units by 50%. Note: When benzoyl-L-arginine ethyl ester (BAEE) is used as the substrate, one BAPNA unit is approximately 45 BAEE umolar units at pH 8.0 at 25°C or approximately 9000 BAEE A253 units at pH 7.6 at 25°C.

One Inhibitor Unit - 1500 KIU

One TIU = 900 KIU

One TIU = 8800 Schwert and Takenake units (STU)

Solubility: Soluble in water (10 mg/ml - completely soluble) and in aqueous buffers of low ionic strengths.⁸ Dilute solutions are generally less stable than concentrated ones. Solution stability also depends on pH; values of 1-12 can be tolerated.¹⁸ Repeated freeze-thaw cycles should be avoided. Sterilization of solutions can be done by filtration through a 0.2 um filter.