LACTASE pH OPTIMUM
Enzymatic activity is strongly dependant on protein conformation. Since pH determines whether an amino acid's side chain is charged or not, and ionic interactions affect tertiary protein structure, pH has a pronounced effect on a protein's conformation and therefore on its enzymatic rate. Typically, the maximum rate of action of an enzyme is found only when it is folded in a precise fashion. The pH which produces this precise folding is termed its pH optimum. An enzyme's pH optimum may be determined by performing multiple assays, each identical except for the pH at which it is run. Graphic display of the resulting data (rate versus pH) demonstrates the enzyme's pH optimum. Here we will determine the pH optimum of the enzyme lactase.
In the preparatory stage of this experiment, an array of buffers have been formulated which cover the pH range to be tested. Typically this can be done by preparing two stock buffers (one acidic, the other basic) which, when mixed together in varying proportions, yield varying pHs. The two stock buffers which we will use are: 1) boric acid/citric acid and 2) Na3PO4. Varying their ratios produces pHs ranging from 2 to 9. For the preparation of these buffers and their proportions for desired pH, see Chemical Technicians' Ready Reference Handbook, p. 656-657.
As in many enzyme assays, adjustments in concentrations and volumes may be needed for optimum results. Keep careful track of how you set up your experiment.
|Materials (per team of two students)||equipment|
|Enzyme dilution for ten tubes
(final conc = 0.625u/mL made by adding 0.1 mL lactase susp'n (9000 units/100 mL) into 14.3 mL dH2O)
20 mM o-nitrophenyl--D galactoside (3.0 mL ONPG)
series of buffers of noted pH made from
boric acid/citric acid + Na3PO4 varying ratios
|test tubes: 10 clean 13x100 mm in rack
displacement pipetters, 0.2 & 1.0 mL
repeater pipetter, 10 mL chamber
37C hot block, 13 mm holes
cuvettes in rack at spectrophotometer
mL 0.1 M pH of mL 20 mM final
tube: buffer buffer dil enz ONPG volume A450 :
B 1.8 dH2O --- 0.2 2.0
1 1.0 2.0 0.80 0.2 2.0
2 1.0 3.0 0.80 0.2 2.0
3 1.0 4.0 0.80 0.2 2.0
4 1.0 5.0 0.80 0.2 2.0
5 1.0 6.0 0.80 0.2 2.0
7 1.0 7.0 0.80 0.2 2.0
8 1.0 7.5 0.80 0.2 2.0
9 1.0 8.0 0.80 0.2 2.0
10 1.0 9.0 0.80 0.2 2.0
2. Add enzyme dilution (0.8 mL) of down the side of each tube, using repeat pipetter, vortex holding tube at the top to wash down the sides.
3. Pre-warm these tubes in a 37C hot block for two minutes.
4. Start the reaction at 15 second intervals: add 0.2 mL ONPG, vortex, start a stopwatch with 1st tube, replace tubes in 37C hot block.
5. Stop the reaction after exactly 15 minutes by adding 1.0 mL 4% K2CO3 down the side of the first tube, mix and remove from hot block. At 15 second intervals, repeat 4% K2CO3 addition for each of the successive tubes, mix and transfer to the test tube rack.
6. Read the absorbency at 450 nm, record in your notebook, graph and discuss results.