Determination of the Trypsin with Potentiometric Method

Determination of the Trypsin with Potentiometric Method

Equipment
Potentiometer = 751 GPD Titrino / Titroprocessor
Electrodes = 6.0234.100 combined micro pH glass electrode; input 1
6.1110.100 T sensor

Reagents

c(NaOH) = 0.1 mol/L in a 1 mL exchange unit; D0

Sample
10 mL borate buffer solution pH = 8.0
1 mL substrate solution
0.05 mL enzyme solution

Remarks

Trypsin is an enzyme which is determined using a TIP method: The pH is adjusted to 8.00 with a pretitration (TrypsPre), then the sample is added and the pH kept at 8 during 8 minutes (TrypsDet). The determination is carried out in a thermostated titration vessel (e.g.6.9914.023 with 6.2036.000 holding ring) at 25 °C.

Calculations:

FIP = calculation using the final volume, according to FIP. Result in FIP units/mg.
FIP(reg) = calculation using the mean rate. Result in FIP units/mg. *)
C01 = (factor for mLL) * (concentration of reagent)(100)
C02 = determination time = 8 minutes
C70 = final volume from TrypsDet
C71 = mean rate from TrypsDet

*) The calculation using the mean rate is fundamentally different form the FIP method (final volume / time). The mean rate is calculated by linear regression over all measuring points of the list. If you wish to use only the linear part of the curve for the regression, use a start time of app. 30 s in the method “TrypsDet“. If one of the formulas is not used, you may delete it.

Sample preparation:

Dissolve the enzyme in c(HCl) = 0.001 mol/L. The solution should not contain more than 50 FIP Units per mL.

Substrate solution:

Dissolve 171.3 mg N-Benzoyl-L-arginine ethylester hydrochloride in dist. water and fill up to 25 mL.

Borate buffer solution pH = 8.00:
Dissolve 286.0 mg Disodiumtetraborate-decahydrate and 1.47 g Calciumchloride-dihydrate in 400 mL dist. water, set the pH with c(HCl) = 1 mol/L to 8.00 and fill up with dist. water to 500 mL.

Comments to parameters:

TrypsDetMonitoring of pH and temperature is ON: pH should be kept in the range of 0.1, the temperature in 0.1°C. Trypsin T may be measured with .

Literature
B. Stellmach, Bestimmungsmethoden Enzyme, Steinkopff Verlag Darmstadt, 1988, p. 263

Method documentation

'pa
751 GPD Titrino 15215 751.0010
TIP Trypsin
parameters
>sequence
1.stirrer:… ON
2.method: …TrypsPre
3.info Add smpl & start
4.method: …TrypsDet
>statistics
status: …OFF
>preselections
req.ident:…. OFF
req.smpl size:… OFF
meas.mode: ….T
temperature ….25.0 °C
------------
'fm
751 GPD Titrino 15215 751.0010
TIP Trypsin
>calculations

FIP=C70*C01/C02/C00;1;U/mg
FIP(reg)=C71*C01/C00;1;U/mg

C00= 0.0507
C01= 100
C02= 8
C70= 0.1920
C71= 0.0238
------------
'fr
751 GPD Titrino 15215 751.0010
TIP Trypsin
smpl size 0.0507 g
C70 0.1920
C71 0.0238
FIP 47.3 U/mg
FIP(reg) 46.9 U/mg
TIP terminated
============