Enzymatic Assay of Aprotinin

1. Objective

To standardize a procedure for the enzymatic assay of Aprotinin.

2. Scope

The scope of this procedure is all products that have a specification for Aprotinin activity.

3. Definitions

3.1 One Trypsin Inhibitor Unit (TIU) will decrease the activity of two Trypsin units by 50% where one Trypsin unit will hydrolyze 1.0 μmole of Nα-Benzoyl-DL-Arginine-p-Nitroanilide (BAPNA) per minute at pH 7.8 at 25ºC.

3.2 Purified Water = water from a deionizing system, resistivity ~18MΩ•cm @ 25ºC

4. Discussion

Aprotinin will inhibit the following reaction:

Nα-Benzoyl-DL-Arginine-p-Nitroanilide    Trypsin   > Nα-Benzoyl-DL-Arginine + p-Nitroaniline

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS

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

7.2 METHOD
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS

7.3.1   200 mM Triethanolamine Buffer, 20 mM Calcium Chloride, pH 7.8 at 25°C (Buffer)
Prepare a 37.1 mg/ml solution in purified water using Triethanolamine Hydrochloride, such as Sigma-Aldrich Product Number T1502, and Calcium Chloride, Dihydrate, such as Sigma-Aldrich Product Number C3881. Adjust to pH 7.8 at 25°C with 1 M NaOH.

7.3.2   0.1% (w/v) Nα-Benzoyl-DL-Arginine-p-Nitroanilide Solution (BAPNA)
Prepare a 1.0 mg/ml solution in purified water using Nα-Benzoyl-DL-Arginine-p-Nitroanilide, such as Sigma-Aldrich Product Number B4875.

7.3.2.1   Keep solution at 25°C prior and during use to ensure a core reaction mixture of 25°C during the run.

7.3.2.2   If the solution is hazy, continue to stir over gentle heat, but do not exceed 65ºC, until the solution becomes clear. Do not use the solution if it turns yellow, this could be an indication of possible chemical decomposition of the substrate due to overheating.

7.3.3   1 mM Hydrochloric Acid Solution (HCl)
Prepare 100 ml in purified water using, for example, Sigma-Aldrich Product Number H3162.

7.3.4   Trypsin Enzyme Solution (Trypsin)
Immediately before use, prepare a solution containing 1040-1560 units/mL of Trypsin, Type III, Sigma-Aldrich Product Number T4665 or equivalent, in cold Reagent 7.3.4 (HCl).

7.3.4.1   The uninhibited rate must have a ∆A405nm/min of 0.08-0.12. Adjust the concentration of Reagent 7.3.4 (Trypsin) using cold Reagent 7.3.4 (HCl) so that the results reside within this range.

7.3.5   0.85% (w/v) Sodium Chloride Solution (NaCl)
Use neat Sigma-Aldrich Product Number S0817 or equivalent.

7.3.6   Aprotinin Inhibitor Solution (Inhibitor)
Immediately before use, prepare a solution containing 0.065-0.080 TIU/ml in cold Reagent 7.3.5 (NaCl).

7.3.6.1   The % inhibition must be between 40 and 60 percent for the assay to be valid. Adjust the concentration of Reagent 7.3.6 (Inhibitor) using cold Reagent 7.3.5 (NaCl) so that the results reside within this range.

7.4 TEST METHOD

7.4.1   Pipet (in milliliters) the following reagents into suitable cuvettes:

    Uninhibited Inhibited
  Blank Test Test
Reagent 7.3.1 (Buffer) 1.600 1.600 1.600
Reagent 7.3.3 (HCl) 0.200 ---- ----
Reagent 7.3.4 (Trypsin) ---- 0.200 0.200
Reagent 7.3.5 (NaCl) 0.200 0.200 ----
Reagent 7.3.6 (Inhibitor) ---- ---- 0.200

7.4.3   Mix by inversion and equilibrate to 25°C. Monitor the A405nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.2 (BAPNA) 1.000 1.000 1.000

7.4.3   Immediately mix by inversion and record the increase in A405nm for approximately 5 minutes. Obtain the ∆ A405nm/Minute using the maximum linear rate for the Uninhibited and then calculate the Inhibited, and Blank Solutions using the same time frame as the uninhibited.

7.5 CALCULATIONS

TIU/ml = (ΔA405nm/minute Unihibited - ΔA405nm/minute Inhibited)(df)
  (9.96)(mL Aprotinin/mL RM)


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

TIU/mg solid = TIU/ml
  mg solid/ml

 

% Inhibition = (ΔA405nm/minute Unihibited - ΔA405nm/minute Inhibited) X 100
  (ΔA405nm/minute Unihibited - ΔA405nm/minute Blank)

7.6 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) Nα-Benzoyl-DL-Arginine p-Nitroanilide, 0.07 mM Hydrochloric Acid, 208-312 units of Trypsin, 0.057% (w/v) Sodium Chloride, and 0.013 – 0.015 Trypsin Inhibitor Units of Aprotinin.

7.7 NOTES

7.3.1   All product and stock numbers, unless otherwise indicated, are Sigma product and stock numbers.

8. References & Attachments

8.1 Fritz, H., Hartwich, G., Werle, E., Hoppe-Seylers Zeitschrift Für Physiologishche Chemie (Berlin) 345, 150-167 (1966).

8.2 Kassell, B., Methods in Enzymology XIX, 844-852 (1970).

8.3 Replaces PROC-DEK-OP-002996 (v1)

9. Approval

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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