Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES UNIVERSITY | METHODS | KALLIKREIN-LIKE ACTIVITY


 

Determination of kallikrein-like activity in plasma with Chromogenic Substrate S-2302

Measurement Principle
The plasma kallikrein-like activity catalyses the splitting of p-nitroaniline (pNA) from the substrate H-D-Pro-Phe-Arg-pNA (S-2302). The rate at which the pNA is released is measured photometrically at 405 nm. This can conveniently be read after stopping the reaction with acetic acid (acid stopped method).The activity measured is mainly the kallikrein-a2- macroglobulin complex.

H-D-Pro-Phe-Arg-pNA + H2O Kallikrein-like activity
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 20 ml of distilled water.
  2. Tris Buffer, pH 7.8 (25C)
    Tris 6.1 g (50 mmol/l)
    NaCl 6.6 g (113 mmol/l)
    Distilled water 800 ml  

    Adjust the pH to 7.8 at 25C by adding an appropriate amount (approx. 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 to 8C.

  3. Acetic acid, 20%

Specimen collection
Blood (9 vol) is mixed with 0.1 mol/l sodium citrate (1 vol) and centrifuged at 2000 x g for 20 minutes at 15-25C. In order to avoid low-temperature activation of prekallikrein the plasma should be kept at 15-25C for not more than a few hours or immediately frozen at -20C or below. After thawing at 37C the plasma should be kept at 15 to 25C and used as soon as possible. Frozen plasma may loose some kallikrein-like activity on freezing or thawing, but is stable for several months at -20C or below.

Method

Sample dilution

Buffer 1000 ml
Test plasma 100 ml
Mix  

 

Acid stopped method

A

Diluted sample

200 ml

Incubate at 37C

3-4 min

Substrate (37C)

200 ml

Mix and incubate at 37C

10 min

Acetic acid 20%

200 ml

Mix  

Plasma blanks are prepared by adding the reagents in reverse order without incubation. Read the absorbance (A) of the sample against its blank in a photometer at 405 nm. The color is stable for at least 4 hours.

Calculation
Plasma kallikrein-like activity in enzyme activity units:
kat/l = A x 5.73
U/l = A x 344

Notes:

  1. The substrate S-2302 is also sensitive to plasmin. By testing with 2 mmol/l S-2251 it is possible to check whether plasmin is present in the sample. The substrate S-2251 is not sensitive to kallikrein.
  2. If the method is to be used for subtraction of blank activities in the prekallikrein assay, it may be preferable to dilute the plasma as indicated for that assay.

Bibliography

  1. Gallimore MJ et al. Simple chromogenic peptide substrate assays for determining prekallikrein, kallikrein inhibitor and kallikrein-like activity in human plasma. Thromb Res 25, 293-298 (1982).
  2. Lai HS et al. Plasma active renin, inactive renin and kallikrein in patients with dissaminated intravascular coagulation. J Formos Med Assoc 90, 448-451 (1991).
  3. Helili K et al. Dynamics of kallikrein activity in different biological fluids of pregnant anumals. Agents Actions Suppl 38 (Pt 1), 114-120 (1992).
  4. Groth T et al. Contact activation of plasmatic coagulation on polymeric membranes measured by the activity of kallikrein in heparinized plasma. J Biomater Sci Polym Ed 8, 797-807 (1997).