INTRODUCTION. Very few safe enzyme inhibitors are available for beginning students to use without worry to either health or disposal problems. This lab exercise is safe, and, what is more, it would serve as a good sequel to the study of the lac-operon, a different level of control.
PROTOCOL. In this exercise it is necessary that you understand two things right from the start:
- It is very important that you recognize the difference between lactase and lactose. (What do the suffixes -ase and -ose mean?) For purpose of redundancy, "(enz)" will be appended to all references to lactase.
- One of the main tricks in enzymology is to keep the enzyme and the substrates apart until you are ready to begin the reaction.
General Procedure. In this experiment you wish to test various candidate inhibitors, and so you will make two batteries of tubes, and then mix them both - all at the SAME time - when you want to start timing the reactions. When you dump the one set into the other, it is like opening the starting gate at a horse race. Furthermore, the dumping of a larger volume into a small one causes a lot of mixing. But first we need to make the needed --
STOCK SOLUTIONS:
- First you need to make 30 ml each of various 'diluants', which are saturated solutions of several sugars, which should include lactose (LAC), galactose (GAL), and glucose (DEX). Shown here, sucrose (SUC) is an additional sugar that is being tested. (Note: since you are using two sugars with "G" as their first letters, use D or DEX as the alternative because the old name for glucose is dextrose.)
- Next make up your stock solution of enzyme. Add TEN DROPS of liquid Lactaid® per 30 ml of water; mix.
Turning to the EXPERIMENTAL SET-UP:
The circled numbers in the diagram indicate the order of adding components.
- Making Battery 1 (ONPG):
- Label 5 tubes as follows (use the capital letters): W (water), S (sucrose), L (lactose), G (galactose), and D (dextrose = glucose)
- Add 10 drops or 100 μl of ONPG into each tube. That is all!
. - Making Battery 2 (lactase (enz) and diluents):
- In front of Battery 1 place five more similarly labelled tubes.
- Into each tube add 2 ml of lactase (enz) solution
- Into the appropriate tube, add 4 ml of the respective diluent (W, S, L, G or D).
. - Get ready to observe and record results!. Paper out, pencil ready, clock.
. - To START the reactions simultaneously, dump Battery 2 tubes into their respective Battery 1 tubes..
. - Observe and record the order (1 to 5) in which the tubes turn yellow (some may not not become yellow at all)..
. - Discuss within your group three possible molecular mechanisms for the result in each tube, particularly why ones are slower (if any)..
. - Record your data and conclusions on the Q&A Sheet.
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