ENZYME ASSAY: LACTASE  ©David B. Fankhauser, Ph.D. Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
Suspending lactase  in buffer	This page has been accessed  times since 21 October 2003.  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.)]	Lactase assay,  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 to digest lactose as they enter their teens.  As a result, they suffer GI upset when they consume milk products (lactose intolerance). 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)
 

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 1:30: Add 0.2 mL of enzyme suspension into 5.8 mL 0.01 M PO4 in a 16 x 150 mm tube.  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 assay set:           5.6 mL 0.1 M PO4 pH 7 buffer                                                                                      1.4 mL 20 mM ONPG
adding rxn mix with repipeter	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.  (Not the enzyme yet.) tube mL  dH 2O mL  Reaction Mix uL  diluted  enzyme A 450: 1 1.00 1.0 0   2 0.975 1.0 25   3 0.950 1.0 50   4 0.900 1.0 100   5 0.800 1.0 200
	4. Pre-warm these tubes in a 37C 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.
	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: 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

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