Biochemistry

Tuesday, 7 May 2013

19:59 -

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Experiment 3: Experiment on enzyme

TITLE: Experiment On Enzyme

OBJECTIVES:

1. To determine the effect of substrate concentration on amylase.

2. To determine the effect of different temperature on amylase.

3. To determine the effect of different pH on amylase.

INTRODUCTION:

The study of enzymes is an integral part of the biochemistry curriculum in universities. Therefore, it is necessary to familiarize the students with the knowledge of how to develop an enzyme assay and how to observe the effects of various factors on the activity of an enzyme. In this laboratory, we will examine the kinetics of α-amylase as found in saliva. Enzyme reacts differently in different environments. The properties of the environment may influence the rate of an enzyme to react. For example, pH, temperature or substrate concentration. In this lab, we will determine the effect of such to amylase.

PROCEDURES:

A) Preparation of standard reference

1. Prepare starch solutions from the stock solution (1.0 mg/ml) into dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution.

2. Iodine solution is prepared by adding 5 g potassium iodide to 100 ml water. The dissolved potassium iodide is added with 1 g of iodine and is allowed to dissolve.

3. Prepare a standard curve of Absorbance (@ 590 nm) vs. Concentration of a starch/iodine

mixture.

B) The effect of substrate concentration

Experiment of starch hydrolysis in different substrate concentration experiment must be prepared as the following table.

Data analysis

1. Calculate starch concentration for each sample after hydrolysis (SF) through use of the standard curve. The initial starch concentration (S0) is already known. [S] = (So) – (SF)

2. The velocity (rate of digestion) of the reaction for each sample can be

calculated as:

V = Δ S/ Δ t = (S0 - SF) / 10 minutes

3. Prepare a table showing rate of hydrolysis (V) at different the starch

concentrations.

4. Plot a Michaelis-Menten graph.

5. Prepare a graph of 1/starch concentration (x-axis) versus 1/rate of digestion (y-axis). This type of reciprocal graph displaying enzyme kinetics is a Lineweaver-Burke plot.

6. State the value of Vmax and Michaelis constant Km from your graph.

C) The effect of temperature

Prepare as the following for the experiment of different temperature.

Data analysis

Plot the Lineweaver-Burke line for the result of 20, 28, 35 and 40°C.

Compare all three plots.

What are the values of Vmax and Km for all plots?

D) The effect of pH

Prepare the following for the experiment using different pH.

Data analysis

What are the values of V for all pH?

Compare the velocity for each of the pH test

RESULTS:

A) Preparation of standard reference

Graph 1: Standard Curve

A) The effect of substrate concentration

Test tube	So (mg/ml)	Absorbance (nm)	SF (mg/ml)	S = (So - SF)	1/S	V = S/10 minutes	1/V
1	0	0.018	0.005	-0.005	-200	-0.0005	-2000
2	0.01	0.06	0.01	0	0	0	0
3	0.025	0.148	0.03	0	0	0	0
4	0.05	0.279	0.07	-0.02	-50	-0.002	-500
5	0.1	0.472	0.12	-0.02	-50	-0.002	-500
6	0.3	1.245	0.3	0	0	0	0
7	0.5	1.912	0.49	0.01	100	-0.001	1000
8	0.7	2.471	0.61	0.09	11.1	-0.009	111.11
9	1	3.324	0.81	0.19	5.26	0.019	52.6

Graph 2: Michaelis-Menten Graph

Graph 3: Lineweaver-Burke Graph

B) The Effect of Different Temperature

Temperature: 10ºC

Test Tube	So (mg/ml)	Absorbance (nm)	Sf (mg/ml)	S = (So – Sf)	1/S	V = S/20mins	1/V
1	0.000	0.298	0.0275	-0.0275	-36.364	-0.001375	-727.27
2	0.010	0.201	0.0384	-0.0284	-35.211	-0.00142	-704.22
3	0.025	0.204	0.0391	-0.0141	-70.922	-0.000705	-1418.44
4	0.050	0.190	0.0361	0.0139	71.94	0.000695	1438.85
5	0.100	0.213	0.0407	0.0593	16.863	0.002965	337.268
6	0.300	0.172	0.0325	0.2675	3.7383	0.01337	74.766
7	0.500	0.173	0.0326	0.4674	2.1395	0.02337	42.7899
8	0.700	0.204	0.0391	0.6609	1.51308	0.03304	30.2618
9	1.000	0.253	0.0491	0.9509	1.0516	0.0475	21.0327

Temperature: 28ºC

Test Tube	So (mg/ml)	Absorbance (nm)	Sf (mg/ml)	S = (So – Sf)	1/S	V = S/20mins	1/V
1	0.000	0.185	0.0350	-0.035	-28.5	-0.00175	-571.43
2	0.010	0.162	0.0304	-0.0204	-49.02	-0.00102	-980.39
3	0.025	0.154	0.0286	-0.0036	-277.78	-0.00018	-5555.56
4	0.050	0.240	0.0465	0.0035	285.71	0.000175	5714.28
5	0.100	0.131	0.0239	0.0761	13.1406	0.003805	262.81
6	0.300	0.190	0.0361	0.2639	3.7893	0.01319	75.7862
7	0.500	0.209	0.0401	0.4599	2.1744	0.00153	653.595
8	0.700	0.163	0.0306	0.6694	1,4939	0.03347	29.877
9	1.000	0.190	0.0361	0.9639	1.03745	0.04819	20.7490

Temperature: 35ºC

Test Tube	So (mg/ml)	Absorbance (nm)	Sf (mg/ml)	S = (So – Sf)	1/S	V = S/20mins	1/V
1	0.000	0.245	0.0475	-0.0475	-21.0526	-0,002375	-421.05
2	0.010	0.144	0.0267	-0.0167	-59.8802	-0.000835	-1197.606
3	0.025	0.143	0.0265	-0.0015	-666.67	0.00007	-1333.3
4	0.050	0.141	0.0260	0.024	41.667	0.003	1000
5	0.100	0.152	0.0283	0.0717	13.947	0.000855	1169.591
6	0.300	0.192	0.0365	0.2635	3.79506	0.013175	75.90
7	0.500	0.839	0.0675	0.4325	2.3121	0.02162	46.2428
8	0.700	0.161	0.0303	0.6697	1.4932	0.03349	29.6864
9	1.000	0.106	0.0177	0.9823	1.01802	0.04911	20.3604

Temperature: 40ºC

Test Tube	So (mg/ml)	Absorbance (nm)	Sf (mg/ml)	S = (So – Sf)	1/S	V = S/20mins	1/V
1	0.000	0.082	0.014	-0.014	-71.4285	-0.0007	1428.57
2	0.010	0.107	0.019	-0.009	-111.111	0.00045	2222.22
3	0.025	0.110	0.021	0.004	250	0.0002	5000
4	0.050	0.093	0.016	0.034	29.412	0.0017	588.24
5	0.100	0.100	0.018	0.082	12.195	0.0009	1111.11
6	0.300	0.088	0.015	0.285	3.5088	0.00075	1333.33
7	0.500	0.091	0.015	0.485	2.06185	0.00075	1333.33
8	0.700	0.074	0.012	0.688	1.4535	0.0006	1666.66
9	1.000	0.184	0.037	0.963	1.03842	0.04815	20.768

Graph 4: Lineweaver-Burke

Temperature 10°C (V_maxand K_M)
Vmax= 32
Km= -5.80

Temperature 28°C (V_maxand K_M)
Vmax = 32
Km= -2.60

Temperature 35°C (V_maxand K_M)
Vmax = 32
Km= -7.65

Temperature 40°C (V_maxand K_M)
Vmax = 32
Km = -1.00

C) The effect of Different pH value

Test tube	pH	Absorbance	S_o	S_f	S=( S_o- S_f)	V=s/time
1	4	0.840	0.5	0.200	0.300	0.015
2	5	0.432	0.5	0.100	0.400	0.020
3	6	0.247	0.5	0.059	0.441	0.022
4	7	0.208	0.5	0.045	0.455	0.023
5	8	0.564	0.5	0.131	0.369	0.018
6	9	0.108	0.5	0.021	0.479	0.023
7	10	0.061	0.5	0.020	0.480	0.024
blank	water	0.252	0.5	0.059	0.441	0.022

DISCUSSION:

The Effect of Substrate Concentration

Catalysts are agents that speed up chemical processes. Almost all catalysts used by living cells are called enzymes. Enzymes are proteins and each cell produces hundreds of them. Enzymes accelerate the velocity of virtually all reactions that occur in biological systems including those involved in breakdown, synthesis and chemical transfers.

The binding of an enzyme to its substrate is an essential part of the enzyme-catalyzed reaction. As the Lineweaver-Burke graph shown above, at low substrate concentration, the active site on the enzyme is not saturated by substrate and thus the enzyme is not working at full capacity. As the concentration of the substrate increases, the sites are bound to a greater degree until saturation when no more sites are available for substrate binding. At this saturating substrate concentration, the enzyme is working at full capacity and the maximum velocity (V_max) of the reaction is seen as 0.019 and the Km is 0.03. The Km of substrate would indicate that the substrate-binding site would be half saturated when the substrate is present at that concentration.

The consequence of saturating the enzyme with substrate on the reaction rate is shown in Michaelis-Menten graph. The initial velocity of the reaction increases in a hyperbolic manner as the substrate concentration is increased. The increase in reaction rate is proportional to the concentration of the enzyme-substrate complex. Thus, V_max occurs because the enzyme becomes saturated with substrate.

The Effect of Different Temperature oon Enzyme reactions.

Commonly we know that enzymes reaction is mainly influence by the temperature that surrounds the reaction this happen because certain factors that focusing on the molecular level of reactions between the atoms within the enzymes.As the temperature increasing the reaction by increasing the collisions as the atoms receive energy so it will collide often and this also cause the lowering of activation energy dor exergonic reactions.Next,what happen is that the number of collision increase tremendously because in order for the reaction between enzymes and substares to occur they need to collide so that it could bind to the active site.Lastly is that when the temperature is too high the temperature inside the molecule is going to become higher as there are certain kinetic energy contributing to the increase of temperature inside the molecules causing the bonds becomes weak and the enzymes proteins begin to become denatured.During this stage the reaction is no longer increasing but it begin to slow down and eventually stops.

That is what suppose to happen and from this experiment we are going to measure the velocity of the reaction in order to know the rate of reaction influence by the temperature.There are 4 different temperatures which is 10°C.28°C.35°C and 40° C.Based on the results mostall the grapg being plotted using the Lineweaver Burke graph but none showed a good patterns as there are no saturated part as the graph is happen to be in a straight line.Actually if the grapg is good we are able to know the V_maxand the K_m from the grapg itself but based on the grapg we obtained all showed negative resulta because all the reading is zero thus we are unable to really compare the reaction rate based on the velocity for the reaction of enzyme from different temperature.

The effect of Different pH values

The activity of enzymes is also greatly influenced by acidity or alkalinity. Excess acidity or alkalinity generally causes denaturation and inactivation of enzymes. Enzymes have an optimum pH or pH range in which their activity is maximum. At higher or lower pH their activity decreases. The active sites on enzymes are often composed of ionizable groups which must be in the appropriate ionic form to maintain the conformation of the active site, bind the substrates or catalyze the reaction. On the other hand one or more of the substrates may contain ionizable groups and only one ionic form of that substrate may bind to the enzyme or undergo catalysis.

CONCLUSION:

In conclusions,substrate which is higher in reaction rate when substrate concentration is high and when the temperature is rising but both has the satirated point where the reaction could not go any further and eventually stops.As for the pH when the excess acidity or alkalinity actually cause denaturation and inactivation of enzymes so enzymes works best only on certain specific temperature.

REFERENCE:

Enzyme Kinetics (2012). Retrieved on May 2, 2013 from

http://www.columbia.edu/itc/chemistry/chem-c2407/hw/ENZYME_KINETICS.pdf

A look into enzyme kinetics: Some introductory experiments (2010). Retrived on 2 MAy, 2013 from

http://onlinelibrary.wiley.com/doi/10.1016/0307-4412(92)90121-2/pdf

Effects Of Temperature On Enzyme Activity retrieved on May 2,2013 from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

Tuesday, 2 April 2013

06:02 -

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Experiment 2: Protein Experiment

Introduction:

There is no single protein assay method that yields absolutely accurate results. The only true and accurate method for determining protein concentration is by acid hydrolyzing a portion of the sample and then carries out amino acid analysis on the hydrolyzate. But, this method is time-consuming. Each method and assay has its own disadvantage and limitations. In this experiment, Biuret assay and Lowry assay method will be use to determine protein concentration. The spectrophotometer is used to measure the amount of light that a sample absorbs.

Objectives:

1. To determine protein concentration of the unknown protein solution

2. To draw standard curve by plotting the absorbance (nm) against the concentration of protein standard solution (mg/mL).

Materials:

1. Prepared solutions of gelatin at 1, 2, 3, 4, 5, and 6 mg/mL in water à Biuret assay

2. Prepared 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL à Lowry assays

3. Biuret reagent

4. Lowry reagent 1

5. Lowry reagent 2

6. Protein

Procedures:

Burette Assays:

1) Firstly the gelatine solutions were prepared at the concentrations of 1, 2, 3, 4, 5 and 6 mg/ml.

2) Then, we take out only 0.5ml of the protein from 1 mg/ml volumetric flask.

3) Then it was being mixed with 2.5ml of the Burette reagent.

4) After that it was left for 10 minutes and then being measured the absorbance.

5) Other concentrations are being repeated with the same steps.

Lowry Assays:

1) The concentration of the protein was prepared under 50 times dilution which is 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 mg/ml.

2) Next, only 0.25ml of the protein being taking out and put into the test tube.

3) Then, it being mix with another 2.5ml Lowry Reagent 1.

4) After 10 minutes, the mixture being added with the Lowry Reagent 2 and being left for 30 minutes.

5) After the time allocated finish so we could measured the absorbance value.

Results

Sample

Type of Eggs	Absorbance Value(nm)	Concentrations(mg/ml)	Real Value (50x dilutions)(mg/ml)
Telur biasa	0.199	2.5	125
Kampong	0.190	2.3	115
Puyuh	0.175	2.1	105
Itik	0.183	1.9	95
omega	0.162	1.5	75

STANDARD SOLUTION

BIURET ASSAY

Concentration (mg/mL)	Absorbance (nm)
1	0.154
2	0.180
3	0.218
4	0.229
5	0.289
6	0.426

LOWRY ASSAY

Concentration (mg/mL)	Absorbance (nm)
0.1	0.070
0.2	0.125
0.3	0.065
0.4	0.130
0.5	0.108
0.6	0.155

Discussion

Biuret assay and lowry assay is a method used to determine the concentration of protein. Absorbance at 540 nm for the Biuret and 750 nm for the Lowry assays. Both need alkali condition for method to be appropriate. Biuret test detect the presence of peptide bond. Peptide bond occurs with the same frequency per amino acid in the peptide. The intensity of colour is directly proportional with the protein concentration. The spectrophotometer was used to. Measure the intensity of the color produced. When the protein concentration is higher it will turn the colour to darker. The colour changes because biuret reagent was react with the peptide bond. Lowry assay lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteay phosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acids. The Lowry method is sensitive to pH changes so for this method to be appropriate it needs alkali condition. Other than that lowry method is sensitive to low concentrations of protein.

The other method to determine the protein concentration by ultraviolet (UV) absorbance at 280 nm for range 0.1-100 ug/ml and Bradford or Coomassie brilliant blue protein assay for range 20-2000 ug/ml . ultraviolet (UV) absorbance at 280 nm ,Aromatic amino acids tyrosine and tryptophan give proteins the characteristic ultraviolet (UV) absorption spectrum at 280 nm. Phenylalanine and disulfide bonds also contribute the absorption at that wavelength, albeit slightly. This method is quickest but error prone and requires an extremely small sample volume since new spectrophotometers employ a sample retention system during the measurement.The protein sample should be pure and does not contain any non-protein components with the same absorption spectrum, such as the contamination of nucleic acids.

Bradford or Coomassie brilliant blue protein assay ,it relies on the formation of a complex between Coomassie brilliant blue G-250 dye and proteins in solution. The free dye exists in four different ionic forms. The more anionic blue form binds to proteins and has an absorbance at 590 nm. The protein concentration can be evaluated by determining the amount of dye in the blue ionic form and by measuring the absorbance of the solution at 595 nm using a spectrophotometer . The dye binds mostly to arginine, tryptophan, tyrosine, histidine, and phenylalanine residues of the protein. One advantage of this method is its compatibility with reducing agents used to stabilize proteins in solution, which are not compatible with Lowry assay and in some extent with the BCA assay. The limitation of Bradford assay is the incompatibility with low concentration of detergents, which are routinely used to solubilize membrane proteins. , one disadvantage of using BSA is that it exhibits a strong dye response and may under-measure the protein content. Appropriate blank is used for zeroing the spectrometry. It is important to measure absorbance that due to the present of solute of interest

Conclusion

In this experiment, we wished to discover the identities of different types of egg dilution by analyzing their protein concentrations through spectrophotometry. Since the dye we used changed to darker colors when in contact with a higher protein concentration, a higher absorbance meant a higher concentration. From this experiment, we were able to estimate the protein concentrations of our different of types of egg dilution and compare to the standard solution that already have been diluted. For the Biuret assay telur biasa have the highest real value which is 125mg/ml, followed by telur kampung 115mg/ml, telur puyuh 105mg/ml, telur itik 95mg/ml and omega 75mg/ml. However, in Lowry assay we does not obtain the result.