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Asparaginase - Assay

Source:
E. coli
CAS:
9015-68-3
EC:
3.5.1.1

Asparaginase Assay

Method
An assay method using a cationic glass electrode has been reported by Ferguson et al. (1974). Jayaram et al. (1974) report on a spectrophotometric assay and compare it with four other methods. Cooney et al. (1972) report on a colorimetric technique. See also Pajdak and Pajdak (1974 and 1972) Frohwein et al. (1971) and Kojima and Wacker (1969). The assay used at Worthington follows:

Method: Essentially that of Mashburn and Wriston (1963) where the rate of hydrolysis of asparagine is determined by measuring released ammonia. One unit releases one micromole of ammonia per minute at 37°C and pH 8.6 under the specified conditions.

Reagents
  • 0.05 M Tris-HCl buffer pH 8.6
  • 0.01 M L-asparagine in 0.05 M Tris-HCl buffer, pH 8.6
  • 1.5 M Trichloroacetic acid
  • Nessler's Reagent
  • Ammonia Standard - Dissolve 1.179 gm ammonium sulfate to a final volume of 100 ml. Dilute 1.4 ml of this solution to 100 ml to give 1 umole NH3 per ml.
Enzyme

Immediately prior to use, prepare several enzyme samples ranging from 0.1-1.0 mg/ml in 0.2 M Tris-HCl pH 8.6.

Procedure

For each different enzyme dilution pipette into test tubes as follows:

  Test Blank
0.05 M Tris-HCl pH 8.6 0.2 ml 0.2 ml
0.01 M L-asparagine 1.7 ml 1.7 ml
1.5 M TCA ----- 0.1 ml

Incubate at 37°C for 5-6 minutes to achieve temperature equilibration. At zero time and at timed intervals add 0.1 ml diluted enzyme to "Test" and "Blank" tubes. Incubate at 37°C for exactly 10 minutes and stop reaction by adding 0.1 ml of 1.5 M TCA to "Test" tubes only. Clarify by centrifugation and add 0.5 ml of clear supernatant to 7.0 ml reagent grade water. Add 1.0 ml of Nessler's reagent and incubate at room temperature for 10 minutes. Read A480 of "Test" tube versus the respective "Blank." Determine micromoles of ammonia released from an ammonium sulfate standard curve. Standard is run in parallel by adding 0.5 ml of 1 umole solution to 7.0 ml water and 1 ml of 1 umole solution to 6.5 ml reagent grade water and adding 1 ml Nesslers reagent to both.

Calculation