 |
ENZYME ASSAY: LACTASEProfessor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103 |
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in buffer |
27 Sept 1993, rvsd 25 Oct 94, 17 Sept 95, 22 Oct 96, 20 Sept 99, 18 Sept 00 [See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)] |
after incubation |
| 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).
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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 |
EQUIPMENT 25 mL grad cylinder 100 mL grad cylinder test tubes: five 13x100 in rack 200 and 1000 micropipets and tips vortex Eppendorf Repipeter with 10 mL syringe 37 C hot block, 13 mm holes stopwatch spectrophotometer 2 cuvettes in rack at spectrophotometer |
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suspending crushed lactose tablet in buffer |
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. |
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| *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 |
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 adding rxn mix with repipeter Here is how the repeater pipetter is used. |
3. a. Copy
the following
table into your notebook. b. Then set up a series of numbered 13x100 mm test tubes as follows. c. Add water to the tubes first, then RxnMix with the repeater pipetter. (Not the enzyme yet.)
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4. Vortex and pre-warm these tubes in a 37 C hot block for two minutes. | ||||||||||||||||||||||||||||||
 adding enzyme  votex completed assay tube |
5. At 30 second intervals, add listed uL of enzyme, vortex, start a stopwatch with 1st tube, place in 37 C hot block. | ||||||||||||||||||||||||||||||
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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. | ||||||||||||||||||||||||||||||
 pour assay tube into cuvette  read A 450: |
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:
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REAGENTS, MATERIALS
AND
CALCULATIONS FOR LACTASE ASSAY
page 26
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
EQUIPMENT:
mortar and pestle
100 mL graduated cylinder
ice bath
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
13x100 tubes
37C hot block for 13 x
100 mL
2 stopwatches
spectrophotometer, warmed
up
at spectrophotometer:
cuvettes in rack
wipettes
SUPPLIES:
lactase tablets
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
(ONPG)
15 mL 0.1 M PO
4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K
2CO3
