Esther Juárez

Properties of Enzymes and Competitive Inhibitors.

cancer cell

Properties of Enzymes and Competitive Inhibitors.

ESTHER JUAREZ

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 find 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 and Chemicals:

1 flask of pH 7 buffered ortho-Nitrophenyl-β-galactoside (ONPG)

1 flask of Lactose 8%

1 flask of pH 7 buffered

1 flask of 8% β-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.

  1. 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.

  1. 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.

  1. 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.

  1. 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.

  1. Cover each of the tubes with parafilm and place the tubes in the 37 °C waterbath for 30 minutes.
  1. After 30 minutes, determine if the reaction has occurred in each tube, and notice change in color.
  1. 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

 β-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

1

1

0

1

3

This table represents the total amounts of each solution added to each test tube in order to get 3 mls for each test tube. This table is used only to represent how the result will look like.

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

This graph depicts the contents inside the test tubes after mixing the mentioned solutions.

 

Measurement of O-nitrophenol. (ONPG)

 

Although the appearance of yellow in the tubes indicated that O-nitrophenol was present, the color, alone, did not tell us how much was present. It was possible to measure the amount of O-nitrophenol present by measuring the intensity of the yellow with a spectrophotometer.

  1. The contents of the 5 tubes were poured into spec 20 tubes. The positions were labeled, but the spec tubes were left clear in order to have an accurate measurement absorbance.
  1. Test tube E acted as the control tube for this, since that tube did not contain inhibitor.

Note: Absorbance 420nm in this experiment will be a measure of the concentration of the

O-nitrophenol molecules in each of the solutions.

Using the Spectrophotometer

 

The spectrophotometer was an instrument designed to measure the amount of light transmitted through solutions, or absorbed by substances in the solution. Light of a specific wavelength is emitted from a special bulb and passed through a tube containing a substance solution. The greater concentration of those particles; the greater the absorbance. It is very important to select the most appropriate wavelength of light for use.

These procedures were followed in order to set up the Spectrophotometer.

  1. 420 nm was the wavelength to use in the inhibitor experiment lab designed because

O-nitrophenol maximally absorbs a light at 420.

  1. The Spectrophotometer was zeroed out with the control knob so that the needle reads 0% transmittance on the upper scale.
  2. The control tube A was put in the holder, and the lid was closed. The light control knob was adjusted so that the needle could read 100% transmittance.
  3. The control tube was removed from the holder. The lid was then closed noticing the needle again read 0% transmittance.
  4. All other test tubes were placed into the Spectrophotometer and read as well.
  1. Data for these results was recorded on the following table.

Table 2. Effect of competitive inhibitor concentration lactose on the production of O-nitrophenol.

Effect of Competitive Inhibitor Concentration on production of ONGP Product

Tube

Inhibitor Concentration

Intensity of yellow

Absorbance

μmoles  of ONPG produced/30min

μmoles  of ONPG produced/min

A

0%

++++

1.55

38.75

1.291666667

B

2%

+++

0.43

107.5

3.583333333

C

4%

++

0.13

32.5

1.083333333

D

6%

+

0.02

5

0.166666667

E

8%

0

0

0

0

Calculation of μmoles O-nitrophenol produced per minutes.

Ex. Tube A: μmoles  of ONPG produced/30min

Absorbance/0.004= μmoles  of ONPG produced per 30min

0.155           / 0.004= 38.75 μmoles

Ex 2 Tube A: μmoles  of ONPG produced/min

μmoles  of ONPG produced per 30min/ 30min

38.75 /30=1.291666667 μmoles  of ONPG produced/min

From the absorbance data that was measured the O-nitrophenol produced per minute was calculated.

  1. Each μmole of O-nitrophenol produced an absorbance of 0.004. The absorbance measured was divided by 0.004 to determine the number of μmoles produced during the experiment. The values were recorded in table 2, fifth column.
  2. The measurements that were obtained in the fifth column were divided by 30(number of minutes left in the waterbath) to obtain the number of μmoles of O-nitrophenol produced per minute.

Graph 2. Absorbance measurements for inhibitor concentration lactose on the production of O-nitrophenol.

Results

According to the hypothesis that temperature, enzyme concentration, and concentration will affect the properties and functions of the enzymes. The hypothesis was supported because graph and tables express the change in absorbance, and μ-moles produced.

Discussion

The tables were able to depict the result in order to get better and accurate results for this particular experiment. Measurements have to be performed with precaution, making sure the enzyme and the contents are mixed properly and at the same time.

Conclusion

Other molecules can affect enzyme activity. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Activity is also affected by temperature, chemical environment, change in pH, and the concentration of substrate.

 

Thanks for your interest!

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