Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES UNIVERSITY | METHODS | PLASMA KALLIKREIN ACTIVATOR (PKA)


Determination of PKA in albumin and immunoglobulin preparations with Chromogenic Substrate S-2302

Measurement Principle
Prekallikrein (prekininogenase) is activated to kallikrein by prekallikrein activator (PKA). The kallikrein formed catalyses the splitting of p-nitroaniline (pNA) from the chromogenic substrate H-D-Pro- Phe-Arg-pNA (chromogenic substrate S-2302). The rate at which pNA is released is measured photometrically at 405 nm and can be followed on a recorder (initial rate method).
The correlation between the change in absorbance per minute (DA/min) and the prekallikrein activator concentration is linear between 0 and 51 IU/ml of prekallikrein activator.
The concentration of prekallikrein activator is calculated using an international standard.

Prekallikrein PKA
Kallikrein
H-D-Pro-Phe-Arg-pNA + H2O Kallikrein
H-D-Pro-Phe-Arg-OH + pNA

Reagents

  1. Chromogenic Substrate S-2302, 25 mg    Art. No. S820340
    Reconstitute the substrate S-2302 (MW: 611.6) with 6.8 ml of distilled water. Working solution: dilute one volume of the stock solution with nine volumes of the buffer (Reagent 2). The working solution is stable for 8 hours at 20-25C.
  2. Tris Buffer, pH 7.8 (25C)
    Tris 6.1 g (50 mmol/l)
    NaCl 0.7 g (12 mmol/l)
    Distilled water 800 ml  

    Adjust the pH to 7.8 at 25C by adding approximately 38 ml of 1 mol/l HCl. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for six months at 2-8C.

  3. Prekallikrein Activator
    The 1 st International Standard 1984 (NIBSC, 82/530) contains 85 International Units per ampoule. Reconstitute with 1 ml of distilled water. Refer to PJ Kerry et al., Br J Haematol, 345-352, (1985) for more information on the standardization of PKA.
  4. Prekallikrein fraction
    A prekallikrein fraction is prepared according to the chromatography procedure described in the appendix. Check the quality of the prekallikrein according to paragraph J in the appendix before each test run. The prekallikrein solution is stable for at least one year at -70C.

Sample
Albumin and immunoglobulin preparations.
Dilute the sample to a corresponding prekallikrein activator concentration of 10-40 IU/ml.

Standard curve
The 1st International Standard has a PKA concentration of 85 IU/ml and is diluted as indicated in the table below.

PKA
IU/ml
International Standard
ml
Buffer
ml
0 - 1000
10.2 120 880
20.0 235 765
34.9 410 590
50.2 590 410

Method

Initial rate method
Step A for sample and standard Sample Tube No. 1
Sample or standard 25 ml
Prekallikrein 100 ml
Mix and incubate at 37C in capped tubes 45 min
Step B for sample and standard Sample Tube No. 2
Substrate (37C) 1000 ml
Mixture from tube No.1  25 ml
Mix  

Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37C) for measurement of the absorbance change for at least two minutes in a photometer at 405 nm and at 37C. Immunoglobulin may occasionally contain significant kallikrein activities and thus a blank reading is necessary.

Step A for immunoglobulin blank Blank Tube No. 1
Immunoglobulin 25 ml
Buffer (37C) 100 ml
Mix   
Step B for immunoglobulin blank Blank Tube No. 2
Substrate (37C) 1000 ml
Mixture from tube No.1  25 ml
Mix  

Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37C) for measurement of the absorbance change for at least two minutes in a photometer at 405 nm and at 37C.

Calculation
Calculate DA/min. Perform the following calculation for the assay of prekallikrein activator in Immunoglobulin preparations:
DA/min sample -
DA/min blank
Plot
DA/min for the standards against their prekallikrein activator concentration. Calculate the prekallikrein activator concentration of the sample from the established standard curve.

Bibliography

  1. Snape TJ et al. The assay of prekallikrein activator in human blood products. Dev Biol Stand 44, 115-120 (1979).
  2. Kerry PJ et al. Standardisation of prekallikrein activator (PKA): the 1st International Standard for PKA. Br J Haematol 60, 345-352 (1985).
  3. Briseid K et al. Part of prekallikrein removed from human plasma together with IgG-immunoblot experiments and functional tests. Scand J Clin Lab Invest 59, 55-63 (1999).
  4. Briseid K et al. Removal of IgG from normal plasma and plasma from untreated patient active Crohn's disease-effect on levels of contact factors. Scand J Clin Lan Invest 60, 237-45 (2000).
 

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