USSKAR-30-2 Lactase Session#1

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Presentation transcript:

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

Some Characteristics of (some) Enzymes End-product inhibition -ve feedback control of metabolic pathways lactase catalyses lactose  glucose + galactose investigation of feedback inhibition of lactase by galactose is aim of session #2 aim of session #1 is to… learn the assay… …and find Km using Lineweaver-Burk

Some Characteristics of Enzymes Vmax maximum rate of reaction under ‘infinite’ substrate concentrations… …and other things (eg enzyme conc, pH, etc) Km substrate conc  ½ Vmax Combined in Michaelis-Menten Kinetics… v = Vmax * [S] / (Km + [S])

Michaelis-Menten Kinetics We will look at this curve today, esp Km

Michaelis-Menten Kinetics We will be looking at fairly ‘linear’ part not obvious where Km is…

Use Lineweaver-Burk to calculate Km Same data: intercept on x-axis is -1/Km

This Session Work in threes Work out volumes of things to put in cuvettes eg how much 45mM ONPG stock plus how much ‘other stuff’ to make 2mM ONPG in cuvette? always make total of 3000 μl typical standard vol in cuvette-based assays Cstock x Vstock = Cnew x Vnew 45 x Vstock = 2 x 3000 = 133 μl stock (and 3000 –133 of buffer & lactase) Make up ONPG/buffer mixes in cuvettes first use cuvette tray no lactase yet!

This Session Measuring initial enzymic reaction rate over first 30 seconds (possible –ve by galactose) need to be very careful getting timing right… put 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 Do full experiment once for each person need data for write-up Estimate Km using L-B graph before you leave so we know what to do next time

Dilutions Work in threes Work out volumes of things to put in cuvettes eg how much 45mM ONPG stock plus how much ‘other stuff’ to make 2mM ONPG? always make total of 3000 μl typical standard vol in cuvette-based assays Cstock x Vstock = Cnew x Vnew Make up ONPG/buffer mixes in cuvettes first use cuvette tray no lactase yet!