Protocol for Lineweaver-Burk Plot: Lactase Kinetics
A Lineweaver-Burk Plot is a graphical display in which the inverse of the rate of an enzyme is plotted against the inverse of the substrate concentration. A great deal of information about the enzyme can be gained using this graphic device. This treatment of the data allows extrapolation of the enzymatic rate at infinite substrate which is equal to the maximum velocity (Vmax.). The intercept at the 1/[S] axis is equal to -1/Km. It also allows distinction between competitive inhibitors (which have identical 1/v intercepts but non identical 1/[S] intercepts as the uninhibited enzyme) and non-competitive inhibitors (which have differend 1/v, but identical 1/[S] intercepts). The following experiment produces the data necessary for the plotting of a Lineweaver-Burk plot.
Equipment per team of two:
eight labeled clean 13 x 100 mm tubes in rack
37C hotblock, 13 mm holes, preheated
2 x 25 mL beakers (reaction mix and K2 CO3)
125 mL flask with about 30 mL dH2O
pipetters: repeat pipetter (Rxn Mix)
1000 uL (for H 2O, ONPG and K 2CO3 )
200 uL (for ONPG)
250 mL beaker for used tips
spectrophotometer with cuvettes & wipettes
Reaction mix: 1 team 5 teams
dH 2O 1.9 mL 3.8 mL
0.1 M PO 4 buffer, pH 7.0 10.0 mL 50 mL
lactase,30 FCC units/mL 100 uL 500 uL
aliquot out 1.2 mL with repeating pipetter (10 mL syringe, refilled)
3.5 mL 20 mM ONPG (colorless) in 13×100 mm test tube
4% K 2CO3 : 12 mL in 25 mL beaker
Enter this table into your notebook, then number your tubes, set up in a rack, add the ingredients as specified in the steps below. Fill in A 450 and calculations as you work thru expt. You should also set up an additional set with a putative inhibitor (see bottom of page).
- Prepare reaction mix in 25 mL beaker, mix thoroughly. [Or use class prep]
- Add dH2 O to labeled tubes: the volume required to q.s. to a final vol. = 2.0 mL.
- Deliver 1.20 mL of Rxn Mix to each tube.
- Prewarm to 37C for two minutes.
- At regular intervals (30 sec or 1 min), carefully add appropriate volume of ONPG to start reactions, mix, return to 37C. For small volumes of ONPG, take care to follow correct micropipette technique, especially do not dip too far into ONPG reagent, deliver to the side of the reaction tube just at but not touching the surface (avoid the possibility of picking up lactase and taking it back to the ONPG). Wipe pipet tip with wipette to clean between tubes. Vortex the tube to pick up all of the ONPG which may have been deposited on the side of the tube.
- At 15 minutes, add 1 mL of 4% K 2CO3 and mix at the same regular intervals to reaction tubes to halt the reaction. (Each tube should be incubated exactly 15 min.)
- Read A450 for each tube against the blank tube (contains no ONPG).
- Graph on a linear scale to produce a Substrate Saturation Curve. Here is an example of a linear/linear graph of activity versus substrate concentration.
- Fill in the table of the values for the inverses of substrate concentration and of enzyme velocities.
- Graph to produce Lineweaver-Burk plot. What is the V max? What is the K m? Here is an example of a double recipricol Lineweaver-Burke plot of the same data as above.
Inhibition experiment: Add final conc of 25mM glucose (0.5 mL of 1.0 M soln /12 mL Rxn Mx), or 5mM galactose (0.1 mL of 1.0 M soln /12 mL Rxn Mix) to test for possible inhibition of ONPG digestion. What kind of inhibition, if any, is demonstrated? (19.8% = 1 M glucose = 1 M galactose).