Enzyme Assay: Lactase
[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]
Lactose, (milk sugar) is a disaccharide formed from galactose and glucose. Its beta-galactosidic bond must be hydrolyzed to yield its component monosaccharides before they can be absorbed by the body. In humans, the enzyme lactase performs this task. Many individuals lose the ability produce lactase, and therefore “lactose intolarent” after they enter their teens. As a result, they suffer GI upset when they consume milk products (gas, abdominal pain, diarrhea). Lactase tablets may reduce this problem. We will be assaying the lactase content in commercial tablets, and use this enzyme as a typical enzyme in our subsequent studies.
The substrate used in the assay of this enzyme is o-nitrophenyl–D galactoside (ONPG), which, upon hydrolysis of the -galactosidic bond, yields galactose and o-nitrophenol, a yellow compound (absorption max = 450 nm) ( CRC Handbook: #p679, Merck Index, #6541).
Enzyme activity is proportional to the increase in A 450 during incubation.
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. This is often best accomplished by diagramming the procedure .
Materials and equipment: (per team of two students)
SUPPLIES: PER CLASS:
fresh lactase (suspend in buffer) (9000 unit tablet suspended in 90 mL buffer)
20 mM o-nitrophenyl–D galactoside (20 mL ONPG)
0.1 M PO 4 buffer, pH 5.5 (60 mL)
0.01 M PO 4 buffer, pH 5.5 (120 mL)
4% K 2CO3 (100 mL)
Distribute to each table:
20 mL dH2O in small flask
7 mL reaction mix* in 13 x 100 mm tube
1 mL 1:200 diluted enzyme (0.5 units/mL) in 13X 100 mm tubes
7 mL 4% K 2CO3 in 13 x 100 mm tube
25 mL grad cylinder
100 mL grad cylinder
test tubes: five 13×100 in rack
200 and 1000 micropipets and tips
Eppendorf Repipeter with 10 mL syringe
37 C hot block, 13 mm holes
2 cuvettes in rack at spectrophotometer
1. a: Record the brand of lactase, labeled number of units of lactase/tablet and the expiration date.
b: Weigh one lactase tablet, note whether 9,000 FCC or 3,000 FCC units/tablet.
c: Grind in a mortar and pestle until finely ground.
d: Suspend/dissolve to 100 units/mL: Grind a tablet in about 5 mL of chilled 0.01 M PO4 buffer, pH 7.
For 9,000 unit tabs, q.s. to 90 mL with same buffer, including mortar and pestle rinses.
For 3,000 unit tab, q.s. to 30 mL (Solution will be cloudy because of undissolved binder.)
e: Dilute 1:200: Add 0.1 mL of enzyme suspension into 19.9 mL 0.01 M PO4, pH 5.5, in a 25 mL grad cylinder.
Most enzymes should be kept on ice until ready to use, this may not be necessary for lactase.
*2. Prepare reaction mix (Rxn Mix): Per desk: Per class of 20:
0.1 M PO4 pH 5.5 buffer 5.6 mL 56 mL
20 mM ONPG 1.4 mL 14 mL
3. a. Copy the following table into your notebook.
b. Then set up a series of numbered 13×100 mm test tubes as follows.
c. Add water to the tubes first, then RxnMix with the repeater pipetter. (Not the enzyme yet
4. Vortex and pre-warm these tubes in a 37 C hot block for two minutes.
5. At 30 second intervals, add listed uL of enzyme, vortex, start a stopwatch with 1st tube, place in 37 C hot block.
6. After exactly 15 minutes, add 1.0 mL 4% K2 CO3 down the side of the first tube, mix and remove from hot block. At 30 second intervals, repeat 4% K2 CO3 addition for each of the successive tubes, mix and set aside.
7. Read the absorbency at 450 nm, record in your notebook table, graph and discuss results.
8. Calculate the number of units of lactase (1.000 OD unit/15 min) in the original tablet. (See following protocol). Compare with other brands of lactase.
CALCULATION OF LACTASE ACTIVITY/TABLET:
If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:
units/tablet = A 450 x 100 mL/tablet x dilution factor x 1/(aliquot in mL
REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY page 26
19 September 1993. rvsd 25 October 1994, 18 Sept 95, 20 Sept. ’96
0.1 M PO 4 pH 7.0 BUFFER:
For 200 mL, weigh out: 1.0 g KH2PO 4
1.8 g Na 2HPO4
dissolve in 200 mL H 2O, check pH, adjust to 7.0 if nec. with either H 3PO4 or NaOH. Store at 4C.
0.01 M PO 4 pH 7.0 BUFFER : (for suspension and dilution of enzyme)
Q.s. 50 mL of pH 7.0 0.1 M PO 4 buffer to 500 mL with dH 2O.
20 mM o-nitrophenyl–D galactoside (ONPG): (chromogenic substrate)
Weigh out: 602 mg ONPG
dissolve in about 80 mL 0.01 M PO4 buffer, pH 7.0 with swirling and slight warming. q.s. with buffer to 100.0 mL.]
REAGENT TO HALT REACTION:
4% K 2CO3 : dissolve 8 g K 2CO3 in 200 mL dH2 O, stir to dissolve.
MATERIALS AND EQUIPMENT for team of four assaying given brand of lactase:
(two sub teams each perform an assay) 10/25/94, rvsd 18 Sept ’95, 20 Sept. ’96
mortar and pestle
100 mL graduated cylinder
5.0 mL pipet (for dH 2O)
pipet bulb or helper
2 x 200 lambda micropipettes
(for ONPG and enzyme)
2 x 1000 lambda micropipettes
(for buffer and 4% K2CO3 )
2 16 x 150 mm test tubes
10 13 x 100 mm tubes
two test tube racks for 13×100 tubes
37C hot block for 13 x 100 mL
spectrophotometer, warmed up
cuvettes in rack
100 mL 0.01 M PO 4 buffer
(to suspend and dilute enzyme)
30 mL dH 2O in 125 mL flask
(to make up assay set)
3 mL 20 mM o-nitrophenyl–D galactoside
15 mL 0.1 M PO 4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K 2CO