BISC220/S14: Mod 1 Lab 4

Determination of the Specific Activity of β-Galactosidase

Specific Activity
The purification of an enzyme is an attempt to enrich the extract for the desired enzyme while eliminating other cellular components, notably other proteins. One measure of the success of a purification step can be obtained by assaying the activity of the desired enzyme at saturating substrate concentration relative to the total amount of protein present. Specific Activity of an enzyme (also sometimes referred to as maximum velocity, V_max) is defined as the amount of product formed/unit time (enzyme activity) per milligram (mg) of protein. An enzyme’s specific activity can be employed to evaluate the relative purity of fractions obtained during the purification. In order to evaluate the success of your purification of β-galactosidase, you must measure both the amount of β-galactosidase activity and the total amount of protein (mg) present in the starting material and in your final purified product.

Specific Activity = amount of product formed/unit time/mg protein

β-galactosidase specific activity is often expressed as µmoles of product (ONP) formed per minute per mg of protein.

Specific Activity of β-galactosidase = µmolesONP/minute/mg protein

Once you know the specific activity of your crude extract and your purified fraction, you can proceed to calculate other values useful in determining the success of a purification step such as:

1. total activity = (specific activity) x (total mg protein in preparation)
2. % yield – the amount of protein of interest retained in the purified fraction
   

            = (total activity of the purified fraction/total activity of the starting material (crude extract)) * 100
      3. purification factor – the fold increase of protein of interest in the purified fraction compared to the crude extract
            = (specific activity of the purified fraction/specific activity of the starting material (crude extract))
Table I shows the results obtained during a β-galactosidase purification by researchers, Wallenfels et al. (1959), working with β-galactosidase. The inverse relationship between total activity and specific activity is clear. The yield (12%) was reasonable and the enzyme was purified substantially (13.5 fold).

References:
Kagedal L (1998) “Immobilized Metal Ion Affinity Chromatography” In Protein Purification 2nd ed. (Janson J-C and Ryden L eds) Wiley-Liss, New York.

Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598-599.

Porath J, Olin B (1983) Immobilized metal ion affinity adsoprtion and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22: 1621-1630.

Wallenfels K, Zarnitz ML Laule G, Bender H, Keser M (1959) Biochem Z 331: 459.

Determining the amount of β-Galactosidase in the Total Protein by β-Galactosidase Enzyme Activity Using A₄₂₀ measurement of ONP production

Microsoft Word File: Media:Determining the amount of β gal by enzyme activity.doc

In-vivo, β-galactosidase cleaves lactose to yield galactose and glucose, but in vitro, the appearance of the products of galactose and glucose are difficult to monitor. The colorless compound ortho-nitro-phenyl-galactoside (ONPG) is substituted for lactose and yields upon hydrolysis (cleavage) by β-galactosidase a yellow compound, ortho-nitrophenol, (ONP) and galactose. The addition of concentrated sodium carbonate (Na2CO3) shifts the pH to a very basic pH 11, a condition which inactivates the enzyme. The amount of colored product (ONP), formed from the colorless substrate (ONPG), can be quantified using a spectrophotometer and then converted to a concentration of ONP using its molar extinction coefficient ( Enzyme Specific Activity or Velocity for a sample calculation).

The total protein assay you performed has allowed you to determine the protein concentration of your two fractions (the crude extract and the purified fraction) but you do not know how much of that protein is our enzyme of interest, beta-galactosidase. You will now perform a specific β-galactosidase activity assay on a series of dilutions of both the crude extract (CE) and of the purified fraction (PF) in order to determine the concentration of enzyme that will yield an absorbance in the most accurate range of measurement of the spectrophotometers (0.1-1.0 for the Hitachi spec). Our goal is to find a diluted form of β-galactosidase in the CE & in the PF that gives an absorbance reading of close to 0.5A in the specific activity assay.

Suggestions for appropriate dilutions based on previous experimentation are: CE: 1:25; 1:50; 1:100; 1:200. Since there is more concentrated beta-galactosidase in the PF, dilutions of: 1:50, 1:100; 1:200; and 1:400 should be tested.

Making a serial dilution of the CE and PF for the SA assay:
Since you have a limited volume of both crude extract and purified fractions and you will need a minimum of 100 µl for each assay, it is advisable to make a little more than you need of each dilution but not so much more that you waste your fractions. It is perfectly acceptable, and often preferable, to use part of a stronger concentration to make the next weaker one.

For example, let's assume you want to end up with 250 µl of each dilution. You could start by making 500 μL of the 1:25 dilution of the CE fraction from last week. After preparing 500 µl of a 1:25 dilution and mixing well you could use 250µl of that 1:25 dilution added to an equal volume of buffer to make 500µl of a 1:50 dilution. If you wanted to continue to dilute with buffer equal volumes of each sequentially weaker concentration, you would end up with 250µl of each of the concentrations desired: 1:25, 1:50, 1:100, 1:200. This is a serial dilution.

How would you go about making 250 μL of each of the PF dilutions you want 1:50, 1:100, 1:200 and 1:400? Please show your dilution strategy to your instructor before you proceed.

After your instructor has approved your dilution strategy and you have on ice all of your labeled microfuge tubes with each of the specified dilutions of the CE and PF in Z-buffer (60mM Na₂HPO₄, 60mM NaH₂PO₄, 1mM MgSO₄, 0.27% beta-mercaptoethanol), you are now ready to start the assay.

Protocol for Assay of Specific Activity (at saturating substrate concentration)

1. Label a set of 8 glass tubes with the 4 dilutions of CE and the 4 dilutions of PF to be tested. For the CE, you will test: 1:25, 1:50, 1:100, 1:200. For the PF, test: 1:50. 1:100, 1:200, 1:400. Label tube 9 "BLANK" as a reagent blanks for the spectrophotometer.
2. Pipet 1.9ml of Z-buffer into the 8 glass test tubes prepared above. Pipet 2ml Z-buffer into the 2 reagent blank (tube 9).
3. Pipet 100μl of the appropriate enzyme dilution into the labeled tube containing Z-buffer prepared in #1. Mix well by vortexing. Put NO enzyme in the blanks!
4. Equilibrate all 10 tubes to 28C in a water bath. Five minutes should be sufficient.
5. Start the reaction by adding 400 µl (0.4 ml) of substrate (ONPG, 4mg/ml) to the first tube, vortex immediately, and quickly return the tube to the water bath. In order to insure that all reactions occur for exactly the same amount of time, add 400µl (0.4 ml) substrate at carefully timed intervals, such as every 20 or 30 seconds. What is the effective concentration of ONPG?
6. At exactly 5 minutes after adding ONPG to the first tube, start adding 1000µl (1 ml) of stop buffer, 1M Na₂CO₃ to each tube in the same order at the same time interval. What is the effective concentration of stop buffer? Mix well after each addition. Since you will be calculating specific activity of beta-galactosidase as µmoles of product formed (ONP)/ minute/mg of total protein, timing of the reaction is critical.
7. Pour some of each tube into a set of labeled cuvettes. Make sure the cuvettes are 2/3 to ¾ full. It doesn’t matter if each has exactly the same volume.
8. Read A₄₂₀ in the spectrophotometer. Don’t forget to change the wavelength from 595nm to 420nm. Zero the instrument using the reagent blanks.
9. Calculate specific activity from absorbance using the Beer-Lambert formula. The molar extinction co-efficient of ONP is 4800 M-1 cm-1 and the path length of the cuvette used is 1 cm. The concentrations and total protein content in each of your fractions were determined by the Bradford dye assay.

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