Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES | METHODS | KALLIKREIN INHIBITOR ACTIVITY


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

Measurement Principle
Plasma is incubated with a purified plasma kallikrein preparation. The amount of kallikrein inhibited is proportional to the activity of the kallikrein inhibitor present in the plasma. The remaining amount of kallikrein activity is then determined by using the chromogenic substrate H-D-Pro-Phe-Arg-pNA (chromogenic substrate S-2302). The rate at which p-nitroaniline (pNA) is released is measured photometrically at 405 nm. This can be followed on a recorder (initial rate method) or read after stopping the reaction with acetic acid (acid stopped method).

K-Inhibitor + K (excess) [K-Inhibitor K] + K (residual)
H-D-Pro-Phe-Arg-pNA + H2O K (residual)
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. Plasma Kallikrein
    Use purified human plasma kallikrein (refer to Gallimore MJ et al., 1978). Prepare a solution of 1 nkat S-2302 /ml human plasma kallikrein in Tris buffer pH 7.8. 
    1 nkat S-2302 corresponds to 0.06 U or 0.017 PEU (refer to Friberger P et al. 1979).
  3. Tris Buffer, pH 7.8 (25C)
    Tris 6.1 g (50 mmol/l)
    NaCl 21.1 g (361 mmol/l)
    Polybrene 20 mg
    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.

  4. Normal plasma
    Blood samples are taken from at least 10 healthy donors. For the preparation of the samples, refer to the Specimen collection section.
  5. Acetic acid 20%
    Acetic acid is used in the acid-stopped method.

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 plasma kallikrein inhibitor 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 15-25C and used as soon as possible. Frozen plasma may lose some plasma kallikrein inhibitor activity on freezing or thawing, but is stable for several months at -20C or below.

Standard curve
Normal plasma has a kallikrein inhibitor activity of 100% and is diluted according to the table below (see note 1).

K-inhibitor
%

Normal plasma
ml

Buffer
ml

25 100 300
50 200 200
75 300 100
100 400 -

Method

Sample dilution

Tube No. 1

Buffer 1900 ml
Test plasma or standard (see note 1) 100 ml
Mix  

 

Initial rate method

Tube No. 2

Sample from Tube No. 1

200 ml

Incubate at 37C

3-4 min

Plasma kallikrein

200 ml

Mix and incubate at 37C

5 min

Substrate (37C)

200 ml

Mix

 

Transfer sample immediately to a 1 cm siliconised semi-microcuvette (preheated to 37C) for measurement of the absorbance change in a photometer at 405 nm and at 37C.
Calculate
DA/min.

Acid stopped method

Tube No. 2

Sample from Tube No. 1

200 ml

Incubate at 37C

3-4 min

Plasma kallikrein

200 mll

Mix and incubate at 37C

5 min

Substrate (37C)

200 ml

Mix and incubate at 37C

4 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 inhibitor in percentage of normal plasma.
Plot A or
DA/min for the standards against their concentration of kallikrein inhibitor on log-lin graph paper. Read the kallikrein inhibitor value for the corresponding A or DA/min of the unknown test sample from the standard curve.

Plasma Kallikrein inhibitor in enzyme activity units.
In each test series a kallikrein activity determination with buffer instead of sample dilution must be performed. The difference between this activity and the sample activity is then calculated.
Initial rate method:
kat/l = (
DA/min buffer - DA/min sample) x 104 
U/l = (
DA/min buffer - DA/min sample) x 6250 
Acid stopped method:
kat/l = (Abuffer-Asample) x 34.7
U/l = (Abuffer-Asample) x 2080

Notes

  1. A 150% standard is prepared by diluting 300 l normal plasma with 3700 l buffer. A 200% standard is prepared by diluting 100 l normal plasma with 900 l buffer. For 0% use the buffer only (note that the adsorbance to surfaces can result in lower readings when plasma is absent).
  2. It is suggested, that the spontaneous kallikrein activity (a 2-M complex) should be determined in patients in whom the kallikrein system is suspected to be activated. See Determination of Kallikrein-like Activity in Plasma.

Bibliography

  1. Gallimore MJ et al. The purification of human plasma kallikrein with weak plasminogen activator activity. Thromb Res 2, 409-420 (1978).
  2. Friberger P et al. Determination of prekallikrein in plasma by means of a chromogenic tripeptide substrate for plasma kallikrein. In: KININS II. Biochemistry, Pathophysiology and Clinical aspects, Eds Fujii S et al. Plenum Publishing Corp 67-82 (1979).
  3. Gallimore MJ et al: Studies on plasma inhibitors of plasma kallikrein using chromogenic peptide substrate assay. Thromb Res 16, 695-703 (1979).
  4. Aasen AO et al. Studies on components of the plasma kallikrein-kinin system in plasma samples from normal individuals and patients with septic shock. Advances in Shock Research 4. Alan R. Liss Inc., NY. 1-10 (1980).
  5. Walker ID et al. The coagulation, fibrinolytic and plasma kallikrein systems in acute pancreatitis. In Progress in Chemical Fibrinolysis and Thrombolysis. Vol.V. Ed Davidson J F, Nilsson I M & Ĺstedt B. 291-293 (1980).
  6. Gallimore MJ et al. Further studies on components of the plasma kallikrein system in plasma samples from cancer patients and normal individuals. In: Progress in Chemical Fibrinolysis and Thrombolysis. Vol. V. Ed Davidson J F, Nilsson I M & Ĺstedt B p 256-258 (1980).
  7. Gallimore MJ et al. Activation of the coagulation, fibrinolytic and kallikrein-kinin systems during cardiopulmonary bypass applying extreme hemodilution. Vth Int Conf on Synthetic Fibrinolytic and Thrombolytic Agents. Progress in Fibrinolysis. June 17-20, Malm 1980. Abstract 51.
  8. Gallimore MJ & Friberger P: Simple chromogenic peptide substrate assays for determining prekallikrein, kallikrein inhibition and kallikrein like activity in human plasma. Thromb Res 25, 293-298, (1982).
  9. Baskova IP et al. Inhibition of plasma kallikrein Kininase and kinin-like activities of preparation from the medicinal leeches. Thromb Res 67, 721-730 (1992).
 

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