PaCoS Exercise 2a: ‘Lactase’ – 2nd Session

End-product Inhibition of Lactase by Galactose: Can it be used to Assay Galactose?

Working in groups and recording absorbance on the computer...

  • Measure initial rates of reaction of lactase on ONPG in the presence of known concentrations of galactose
  • Measure initial rate of reaction of lactase on ONPG in the presence of an unknown concentration of galactose
  • Repeat this so each member of the group has their own set of data
  • We will not measure spontaneous (ie in absence of enzyme) breakdown of ONPG as it is 0

Solutions provided

  • 0.1M TRIS buffer, pH 7.0
  • 45mM (13.5 mg/ml)ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1M TRIS, pH 7.0
  • Lactase solution (4.33 U/ml; made from Sigma K. lactis, 2600 U/g)
  • 12%galactose (666 mM) in 0.1M TRIS buffer, pH 7.0
  • an ‘unknown’ galactose solution

Setting up the Spectrophotometer

  • Turn on the spectrophotometer and allow it to warm up (minimum of 10 mins)at 420 nm.

Basic Assay Procedure

  • Note that the basic reaction mix comprises 3000 µl of reagents in a cuvette, as follows…
  • ONPG stock (60 µl)
  • Galactose stock + buffer (combined vol of 2840 µl)
  • Lactase solution (100 µl) : to be added at the start of the recording
  • Put a cuvette containing ONPG/Galactose/buffer in the spec, close the lid and press CAL to zero the reading (at 420nm) - this means the reaction in the next step will start at an absorbance reading of 0, assuming no immediate effect of adding the lactase
  • Open the lid of the spectrophotometer
  • Add 100 μL of lactase solution, rapidly mix it with a pastette and start the timer, then close the spectrophotometer lid
  • Note the absorbance (on the spec) after 30 sec (then double it to get rate per min)

Obtaining data for Inhibition by Galactose

  • For the five galactose standards (zero Galactose is a standard!), calculate the volumes of Galactose stock (666 mM) and Tris buffer needed to produce the required cuvette concentrations once all the reagents are present (ie in 3000 µl), and put these in the table below
  • Make up the five ‘standard’ cuvettes with the ONPG, Galactose and buffer (but not the lactase!).
  • For the sixth, ‘unknown’ cuvette put in 2840 µl of the unknown solution instead of a Galactose stock/buffer mix.
  • Carry out the Basic Assay Procedure for each of the cuvettes.
  • Repeat the above so each member of the group has their own set of unique data.

Results

  • If Vs is the ‘spontaneous’ rate (ie in the absence of enzyme) and Vz is the ‘uninhibited’ rate (iein the presence of lactase but absence of galactose) then the percent inhibition for any other initial absorbance rate (V) is given by…
    % inhibition = 100 - ( (V-Vs)/(VZ-Vs) * 100)

But since we know from last week that Vs is 0, this simplifies to…

% inhibition = 100 – (V / VZ * 100)

  • Calculate the percent inhibitions from your recorded data.

666 mMGalactose (µl) / Tris (µl) / 45 mMONPG (µl) / Lactase (µl) / Total
(µl) / Galactose (mM) / Initial Abs rate
Ab U/min / % inhibition
60 / 100 / 3000 / 0 / (VZ) / 0
60 / 100 / 3000 / 22 / (V)
60 / 100 / 3000 / 55 / (V)
60 / 100 / 3000 / 111 / (V)
60 / 100 / 3000 / 166 / (V)
Unknown (µl)
2840 / 0 / 60 / 100 / 3000 / Unknown / (V)