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 (reaction 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) Na 3PO4. Varying their ratios produces pHs ranging from approximately 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. Refer to the protocols on Lactase Assay and Reagents for Lactase Assay.
Materials (per team of two students)
Enzyme dilution for ten tubes:
final concentration = 0.1 units/mL made by
suspending a tablet to make 100 units/mL (9000 units/90 mL)
dilute 0.1 mL 100units/mL into 100 mL dH 2O (1:1000 dilution)
20 mM o -nitrophenyl–D galactoside (3.0 mL ONPG)
series of buffers of noted pH made from boric acid/citric acid + Na3PO 4 in varying ratios
4% K2 CO3
test tubes: 10 clean 13×100 mm in rack
displacement pipetters, 0.2 & 1.0 mL
repeater pipetter, 10 mL chamber, set on 0.8 mL
37C hot block, 13 mm holes
cuvettes in rack
COPY TABLE INTO YOUR BOOK, USE IT TO SET UP THE TUBES:
- Add enzyme dilution (0.8 mL) of down the side of each tube, using repeat pipetter.
- Add specified buffer (1 mL each) to its appropriate tube (You may use the same pipet tip if you progress in sequence up through the buffers for all ten tubes, blowing out and tipping off any clinging droplets. Cross contamination effects should be minimal. Vortex each tube, holding tube near the top to wash down the sides.
- Pre-warm these tubes in a 37 C hot block for two minutes.
- Start the reaction at 15 second intervals: add 0.2 mL ONPG, vortex, start a stopwatch with 1st tube, replace tubes in 37 C hot block.
- Stop the reaction after exactly 15 minutes by adding 1.0 mL 4% K 2CO 3 down the side of the first tube, vortex and remove from hot block. At each 15 second interval, repeat 4% K 2CO3 addition for each of the successive tubes, mix and transfer to the test tube rack.
- Read the absorbency at 450 nm, record in your notebook.
- Graph results (according to proper graphing technique) and discuss.