Properties of Enzymes and Competitive Inhibitors

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Index Page

Abstract…………………………………………………………………………….….…….3
Introduction……………………………………………………………………….….……..3
Materials and Chemicals used…………………………..……………………..…….…..3
Procedures…………………………………………………………………..…...…...…… 4
Tables……………………………………………………………………………………...5-7
Results……..…………………………………………………………………………...……8
Discussion….………………………………………………..………………………...……8
Conclusion…………………………………………………………………………....…….8
Works Cited ………………………………………………………………………………..9

Properties of Enzymes and Competitive Inhibitors.

Abstract: Properties of enzymes were found in this experiment and some other factors, which affect enzyme activity. Enzymes are catalyst; they catalyze very specific reactions. Results relating to the active site of specific enzymes played a big role while performing this experiment. The purpose of this experiment was to fin how inhibitors affect enzyme’s activity by competing for the active site against substrates.

Introduction: Cells have the ability to perform chemical reactions that at normal temperature outside the body proceed too slowly to support life. Cells are able to perform some reactions rapidly because they possess protein catalyst called enzymes. Enzymes are proteins that catalyze (i.e., increase the rates of) chemical reactions. Each enzyme has a unique globular shape, a small portion of which functions as an active site capable of binding to specific reactants or substrates.

It was hypothesized that enzyme concentration, temperature, and inhibitors will affect the properties and abilities of the enzyme.

Materials:
1Wax Marking Pens
150 ml Beakers
3 400 ml Beaker
1 container of parafilm
1 set of 20 spec tubes
1 regular test tube rack
1 small test tube rack
1 box Kimwipes
Eye Droppers
1 thermometer
2-10ml Graduated Cylinders
1 Spectrophotometer
37 °C waterbath with test tube racks

Solutions:
1 flasks of pH 7 buffered ONPG
1 flask of Lactose 8%
1 flask of pH 7 buffered
1 flasks of 8% beta galactosidase

Procedure

1. Obtain five test tubes and label them (i.e. A, B, C, D, E) 2. Using a 10 ml graduated cylinder put:
Note: It is very important to add enzyme last.

1 ml of pH 7 buffered ONPG + No Lactose 8%(0ml) +(1 ml pH buffer) + Enzyme (1ml) solutions into tube A. 0% Lactose.

3. Using a 10 ml graduated cylinder put:

1 ml of pH 7 buffered ONPG + Lactose 8% (.25ml) +(.75ml pH buffer) + Enzyme (1ml) solutions into tube B. 2% Lactose.

4. Using a 10 ml graduated cylinder put:

1 ml of pH 7 buffered ONPG + Lactose 8% (.5ml) +(.5ml pH buffer) + Enzyme (1ml) solutions into tube C. 4% Lactose.

5. Using a 10 ml graduated cylinder put:

1 ml of pH 7 buffered ONPG + Lactose 8% (.75ml) +(.25ml pH buffer) + Enzyme (1ml) solutions into tube D. 6% Lactose.

6. Using a 10 ml graduated cylinder put:

1 ml of pH 7 buffered ONPG + Lactose 8% (1ml) +(0ml pH buffer) + Enzyme (1ml) solutions into tube E. 8% Lactose.

7. Cover each of the tubes with parafilm and place the tubes in the 37 °C waterbath for 30 minutes.

8. After 30 minutes, determine if the reaction has occurred in each tube, and notice change in color.

9. Test tube E acted as our control test tube because no competitive inhibitor was added. Lactose was the competitive inhibitor for this reaction into the test tube.

Note: Because the result on steps 4 and 6 were not accurate for our particular experiment, steps 4 and 6 were performed twice.

The following table and graph express the results after the measurements and mixing.

Table 1. Measurements after mixing the solutions into the test tubes.

Solutions| pH 7 Buffered ONPG (ml)| Lactose 8% (ml)| pH buffer (ml)| Enzyme B-Gal (ml)| Total amount of mls.| Test tube A| 1| 0| 1| 1| 3|
Test tube B| 1| 0.25| 0.75| 1| 3|
Test tube C| 1| 0.5| 0.5| 1| 3|
Test tube D| 1| 0.75| 0.25| 1| 3|
Test tube E...
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