Lactose, (milk sugar) is a disaccharide formed from galactose and glucose.Its B-galactosidic bond must be hydrolyzed to split its component monosaccharides before they can be absorbed by the body.In humans, the enzyme lactase performs this task.Many individuals lose the ability to digest lactose as they enter their teens, and suffer GI upset when they consume milk products.Lactase tablets may reduce this problem.We will be assaying the lactase content in these 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-B-D galactoside (ONPG), which, upon hydrolysis of the B-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 A₄₅₀during 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.

Materials and equipment: (per team of two students)

lactase suspension (1 tablet suspended in 100 mL)

20 mM o-nitrophenyl-B-D galactoside (1.2 mL ONPG)

0.1 M PO₄ buffer, pH 7.0 (6 mL)

0.01 M PO₄ buffer, pH 7.0 (120 mL)

200 uL and 1000 uL micropipets

Eppendorf Repipettor fitted with a 10 mL syringe 

test tubes: one 16x150, five 13x100 in rack

37 C hot block, 13 mm holes

ice bath

stopwatch

4% K₂CO₃

spectrophotometer

cuvettes in rack at spectrophotometer

1.Recordyour brand of lactase and labeled number of units of lactase/tablet. 

Weighone lactase tablet.

Grindin a mortar and pestle until finely ground.

Suspend/dissolvein about 5 mL of chilled 0.01 M PO₄ buffer, pH 7, q.s. to 100 mL with same buffer.(Solution will be cloudy because of undissolved binder.)

(Here are the ingredients necessary for weighing, grinding, and suspending the tablet.)

Dilute0.2 mL of this into 4.8 mL 0.01 M PO₄ buffer in a 16 x 150 mm tube. 

(1:25 dilution)Keep on ice until ready to use.

Here is a blackboard diagram (informal) of the experimental plan.

2.Copy the following table into your notebook.Then set up a series of numbered 13x100 mm test tubes as follows.

mLuL dilutedmL

mLreactionsuspendedfinal

tube:dH₂OmixenzymevolumeA₄₅₀:

10.801.202.0

20.781.2252.0

30.751.2502.0

40.701.21002.0

50.601.02002.0

Prepare sufficient reaction mix for the number of tubes you will run plus one (per tube:1.0 mL buffer and 0.2 mL ONPG 

3.Add specified quantity of water to each tube 

4.Aliquot out 1.2 mL of reaction mix to each tube

5.Pre-warm these tubes in a 37 C hot block for two minutes.

6.At 30 second intervals, add listed uL enzyme, vortex, start a stopwatch with tube 2.

7.After exactly 15 minutes, add 1.0 mL 4% K₂CO₃ down the side of tube 2, mix and remove from hot block.At 30 second intervals, repeat 4% K₂CO₃ addition for each of the successive tubes, mix and set aside.Finally, add 1 mL 4% K₂CO₃. to tube 1 (blank).  Here are the five tube at the end of the assay, reflecting the amount of enzyme present by the intensity of their yellow color

8.Read the absorbency at 450 nm, record in your notebook, graph and discuss results.

9.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.

REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY

19 September 1993. rvsd 25 October 1994, 18 Sept 95, 20 Sept. '96

0.1 M PO₄ pH 7.0 BUFFER:

For 200 mL, weigh out:1.0 g KH₂PO₄

1.8 g Na₂HPO₄

dissolve in 200 mL H₂O, check pH, adjust to 7.0 if nec. with either H₃PO₄ or NaOH.Store at 4EC.

0.01 M PO₄ pH 7.0 BUFFER: (for suspension and dilution of enzyme)

Q.s. 50 mL of pH 7.0 0.1 M PO₄ buffer to 500 mL with dH₂O.

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% K₂CO₃:dissolve 8 g K₂CO₃ in 200 mL dH₂O, 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 dH₂O)

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

37EC 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 PO₄ buffer 

(to suspend and dilute enzyme)

30 mL dH₂O in 125 mL flask 

(to make up assay set)

3 mL 20 mM o-nitrophenyl-?-D galactoside

(ONPG)

15 mL 0.1 M PO₄ buffer, pH 7.0 

(for assay tubes)

15 mL 4% K₂CO₃

CALCULATION OF LACTASEACTIVITY/TABLET:

If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:

units/tablet = A₄₅₀ x 100 mL/tablet x dilution factor x 1/(aliquot in mL)