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

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!

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

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