Assay Procedure for Gamma Glutamyl
Transferase (Ref. No. FGAP023)
Based on that of Orlowski as described by Szasz in which the rate
of increase in absorbance due to release of p-nitroanaline is measured at 405nm and 25°C.
|
g GT |
|
| Gamma-glutamyl-p-nitroanilide +
glycylglycine |
 |
gammaglutamyl-glycylglycine + p - nitroaniline |
Unit Definition
That amount of enzyme which catalyses the liberation of 1 micro
mole p-nitroaniline per minute at 25°C.
Reagents
| A |
0.05M Amino-2-methyl-propan-1.3 diol buffer pH9.3 Dissolve 2.63g AMP [MM106.14] in 400ml distilled H2O, adjust to
pH 9.3 with 1M HCl and then dilute to 500ml. Store on ice.
|
| B |
Substrate Dissolve
12.05mg L-gamma-glutamyl-p-nitroanilide [MM267.24], 69.4mg glycylglycine [MM132.12] and
21.3mg MgCl2.6H2O [MM 203.30] in 10 ml buffer [A] at 30°
C- 40°C with constant stirring.
Check pH 8.2 and A405 [1cm cell] to 0.4 - 0.6 against
buffer [A]. Store at R/T.
|
| C |
Enzyme Dissolve 5mg
enzyme/ml ice-cold buffer[A]. Immediately before assay, dilute solution to yield 0.1-0.2
u/ml ice-cold buffer [3A]. (DA/min 0.046 - 0.08). |
Procedure
l = 405nm
Light Path = 10mm
Cuvette volume = 3.15ml
Sample Volume = 0.15ml
Temperature = 25°C
Into a 10mm quartz cell, pipette the following:
| Substrate [B] |
3.0 ml |
| Equilibrate at 25°C and monitor DA405/min |
|
| Enzyme [C] at zero time |
0.15 ml |
|
3.15 ml |
Record rate of increase in absorbance at 405nm for ± 5 minutes
after initial time lag. Check temperature in cell.
Calculation
|
DA405/min
x 3.15 x dilution |
| u/mg material = |
 |
|
9.62 x 0.15 x mg enzyme/ml original solution |
Reference
Szasz G.: (1974): Methods of Enzymatic
Analysis
Ed. Bergmeyer, H.U. 2 715 |
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes