BISC220/S13: Sample Calculations Lab 2

ENZYME SPECIFIC ACTIVITY OR VELOCITY

Data:
ONPG assay for enzyme specific activity:
      • Let's assume you have used 0.1 ml of a 1:500 dilution of one of your samples
      • The absorbance reading at 420 nm (A) of your reaction tube was 0.450
      • The total volume in the reaction tube when the A420 reading was taken was 3.4 ml
      • Assume the inside diameter (l) of the reaction tube or curvette was 1.0 cm
      • Molar extinction coefficient of ONP (e) = 4800 M^-1 cm^-1

Calculations for the enzyme assay:

      Concentration (in moles/liter) = A/(e)(l)

      A = absorbance
      e= molar extinction coefficient (M^-1 cm^-1)
      l = pathlength (cm)

The concentration of ONP in moles/liter in your reaction tube is given by:

       [ONP] = 0.450/(4800 M^-1cm^-1)(1.0 cm)

       Our example: [ONP] = 0.94 x 10^-4 moles/L

The amount of ONP that was produced per minute in each ml of the reaction mixture in your reaction tube is given by:

       [ONP]/time = (0.94 x 10^-4 moles/L)/5 minutes (our reaction time)

       [ONP]/time = 1.9 x 10^-5 moles/L/min

Expressed in different units (moles --> mmoles and liter --> ml):

       [ONP]/time = 1.9 x 10^-5 mmoles ONP/ml/min

Convert this to µmoles ONP/ml/min:

       [ONP]/time = 0.019 µmoles ONP/ml/min

The ml here refers to the volume in the reaction tube. The total volume in the reaction tube at the time the reaction was measured was 3.4 ml. Therefore, the amount (µmoles) of ONP produced per minute in the total reaction mixture must have been:

       µmoles of ONP/min = (3.4 ml total volume) * 0.019 µmoles/min/ml

       total µmoles of ONP/min = 0.064 µmoles/min

The 3.4 ml of total reaction volume contained only 0.1 ml of enzyme. If the 0.1 ml enzyme volume came from a 1/500 dilution containing 2.5 mg/ml of total protein before it was diluted, then in 0.1 ml of this 1/500 dilution you would have 0.0005 mg of protein.

DO NOT USE THE VALUES 2.5mg/ml or 1/500 IN THE FORMULA BELOW, UNLESS THAT WAS YOUR CALCULATED PROTEIN CONCENTRATION AND THE DILUTION YOU USED.

       (2.5 mg/ml) * (0.1 ml) * (1/500) = 0 .0005 mg protein

Knowing the amount of ONP produced per min and the protein concentration expressed in milligrams, you may calculate the activity or velocity of your sample. This could also be referred to as the specific activity (SA) because in this case, the substrate was saturating:

       SPECIFIC ACTIVITY OR VELOCITY= (0.064 µmoles ONP/min)/0.0005 mg protein

       SPECIFIC ACTIVITY = 128 µmoles ONP produced/min/mg protein