1. USSKAR-30-2 Lactase Session#2
Practice & Communication of Science
USSKAR-30-2
Lactase Session#2

2. Some Characteristics of (some) Enzymes
End-product inhibition
-ve feedback control of metabolic pathways
lactase catalyses lactose  glucose + galactose
can we demonstrate galactose inhibits?
Methodology: measure initial reaction rates…
Substrate is lactose analogue
ONPG  gal + ONP
Fixed enzyme and sub concs
Varying initial gal concs
Expect to see rates decrease as galactose increases

3. This Session Work in threes
Work out volumes of things to put in cuvettes
eg how much 666mM Galactose stock plus how much ‘other stuff’ to make 22mM Galactose?
Galactose + ‘other stuff’  3000 μl
typical standard vol in cuvette-based assays
Cstock x Vstock = Cnew x Vnew
Make up Galactose/ONPG/buffer mixes in cuvettes first
use cuvette tray
no lactase yet!

4. This Session Measuring initial enzymic reaction rate
over first 30 seconds
need to be very careful getting timing right…
put Galactose/ONPG/buffer cuvette in spec
press Cal to zero, lift lid
add lactase, mix, start stopwatch ‘simultaneously’
note Ab(420nm) at 30s, then x2 to get rate per min
don’t forget to also assay the ‘unknown’ Gal !
Do full experiment once for each person
need data for write-up
Estimate % inhibition if time allows

5. Dilutions Work out volumes of things to put in cuvettes
eg how much 666mM Galactose stock plus how much ‘other stuff’ to make 22mM Galactose?
Galactose + ‘other stuff’  3000 μl
typical standard vol in cuvette-based assays
Cstock x Vstock = Cnew x Vnew