BIO 462 EXPERIMENT 3 ENZYMOLOGY PART 1: DETERMINATION OF OPTIMUM PARAMETERS
NAME: NURFADHILAH BINTI JAAFAR STUDENT ID: 2016675256 PROGRAM: AS 246 GROUP: 4B DATE OF THE EXPERIMENT: 5 TH APRIL 2017 DATE OF SUBMISSION: 12 TH APRIL 2017 LECTURER: MADAM AZANI BINTI SALEH
PROCEDURE -
Refer to Biochemistry Laboratory Manual page 15 until 23 -
RESULTS 1. Enzyme Concentration
Cuvette
OD
OD
OD
Mean ± SD
(replicate 1)
(replicate 2)
(replicate 3)
1
0.080
0.079
0.078
0.079 ± 0.001
2
0.144
0.143
0.143
0.143 ± 0.0006
3
0.213
0.214
0.214
0.214 ± 0.0006
4
0.287
0.287
0.288
0.288 ± 0.0006
OD
OD
OD
Mean ± SD
(replicate 1)
(replicate 2)
(replicate 3)
1
0.096
0.099
0.100
0.098± 0.002
2
0.103
0.105
0.107
0.105± 0.002
3
0.094
0.096
0.098
0.096± 0.002
4
0.094
0.094
0.095
0.094± 0.0006
5
0.068
0.070
0.071
0.0700± 0.002
6
0.051
0.053
0.053
0.0520± 0.002
7
0.041
0.042
0.044
0.0420± 0.002
2. Substrate Concentration
Cuvette
3. pH
Cuvette
OD
OD
OD
Mean ± SD
(replicate 1)
(replicate 2)
(replicate 3)
1
0.092
0.087
0.090
0.090± 0.003
2
0.072
0.073
0.072
0.072± 0.0006
3
0.077
0.080
0.070
0.078± 0.005
4
0.072
0.072
0.072
0.072± 0
5
0.070
0.073
0.075
0.075± 0.003
6
0.230
0.232
0.230
0.231± 0.001
DISCUSSIONS This experiment conducted with the use of spectrophotometer to identify the effect of enzyme concentration, substrate concentration on the rate of reaction and to determine the effect of pH on the enzyme activity. The spectrophotometer is a vital machine for researchers and scientist to in studying various substances and elements. The spectrophotometer was calibrated and set to 410 nm. The absorbance values and the transmittance of light for the substance were measured by the photometer at the end of the spectrophotometer machine which can be plotted on a calibration curve. Enzymes are the vital part of life because enzyme controls all metabolisms and metabolic process that occur. The substrate used in this experiment is starch solution while the enzyme used in this experiment is amylase which is specialized to break down one specific type of compound. Enzymes amylase can be found in human saliva and responsible to break down the starches into simple sugars. The result for the effect of enzyme concentration on the rate of reaction and it shows that as the enzyme concentration increases, the rate of reaction also increases and the result of this reaction resulted with a linear graph which continuously increases. In this experiment, the effect of substrate concentration on the rate of reaction also can be observed with the reading from the spectrophotometer. The graph of the absorbance against the starch concentration that we constructed experimentally was not same theoretically. It shows that the reading of the absorbance become lower as the substrate concentration been added which is opposite from theory. Supposedly when the substrate concentration increases, the solution become more saturated, the rate of reaction also increases, but once all the active enzymes are fully occupied by substrates, the reaction reach the maximum rate and would not increases since all the active sites are all occupied. The reaction initiates at faster rate and become slower as there is not enough enzymes to convert the substrates and this can be solved by addition of more enzymes. The relationship between the effects of pH on the enzyme activity is presented by the graph constructed absorbance against the pH buffer. pH buffer used on this experiment are pH3, pH5, pH7, pH9, pH11 and distilled water. The graph experimentally is differing with theoretically as the enzyme activity become slower when the pH buffer increases. When the pH was 3 or 5 the reaction yielded minimum result. Theoretically, if there is an increase in pH, the rate of reaction also increases, until it reaches the peak value and starts to denature. Any small changes in pH can drastically alter the productivity of the enzymes.
Overall, we could only get the accepted result for the graph of the effect of enzyme concentration on the rate of the reaction only. The possible transmission of errors in this experiment might be due to material handling and personal error which contributed to inaccuracy of result data. In this experiment the measurement used was by ‘drops’ of the concentration is inaccurate measures compared to unit volume such as millimeter which can contribute to either predictable or accurate results. Other random error also happen when we used the unclean cuvette, thus the interferences inside the unclean cuvette may react with the enzyme or substrate concentration added. The systematic error may occur when the spectrophotometer was not calibrated properly. Other possible ways to see improvements for this result would have been more replicate to provide much more representation how the curve on the graph actually is. CONCLUSIONS In conclusions, the objective of the experiment to calibrate the spectrophotometer, determine the enzyme concentration, substrate concentration on the rate of reaction and the effect of pH on the enzyme activity was achieved. It shows that the graph become linear when as the amount of the enzyme concentration increases, the rate of reaction also increases. For both graph of the effect of substrate concentration on the rate of reaction and the effect of the pH on the enzyme activity, we could not get the desirable curve as theoretical graph curve which may due to some error. The pH on the enzyme amylase should have an effect and major influence on the enzyme activity as the pH and if the substrates are unlimited in availability, then the rate of reaction of the enzyme also decreases. Overall, it was apparent that change in enzyme concentration, substrate concentration and pH level does have an effect on the enzyme activity and the rate of the reaction.
