Substrate concentration in enzyme reactions
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Our lab has equipped with a UV-visible microplate reader absorbance, providing ultrafast, full spectral range detection for cuvettes, 96-well, and 384-well microplates. We provide services with measurement accuracy and linearity over the widest range of assays, including DNA and protein quantifications, MTT and MTS etc.
Enzyme activity is a measure of how much enzyme is present in a reaction. There are two ways to measure enzyme activity: monitoring the disappearance of substrate or the appearance of product. Measuring the appearance of product is usually more accurate because detecting small changes in [P] (when [P]=0) is easier to measure than detecting small changes in [S].
Through Michaelis-Menten kinetics, enzyme assays are used to calculate the enzyme's Km for a specificsubstrate, Vmax, and Ki for inhibitors. Enzyme assays can also reveal information about the substrates and inhibitors that may affect the enzyme. Figure 2 below uses a fixed-time assay to demonstrate the effect adding enzyme inhibitors have in a reaction. Details of enzyme-substrate reaction rates are further described by the Michaelis-Menten Kinetic Model. |
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OBJECTIVES
To obtain the rate of enzyme reaction
To study the effect of substrate concentration in enzyme reaction
To study the effect of temperature in enzyme reaction
To obtain the rate of enzyme reaction
To study the effect of substrate concentration in enzyme reaction
To study the effect of temperature in enzyme reaction
PROCEDURES
1. Starch solution had been prepared from the stock solution (1.0mg/ml) into dilution 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 ready with added 5g potassium iodide to 100 l water. The potassium iodide that has been dissolved is added with 1g of iodine and is allowed to dissolved.
3. The standard curve of absorbance vs concentration of a starch/ iodine is prepared.
4. The solution of substrate concentration effect and temperature effect also prepared.
1. Starch solution had been prepared from the stock solution (1.0mg/ml) into dilution 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 ready with added 5g potassium iodide to 100 l water. The potassium iodide that has been dissolved is added with 1g of iodine and is allowed to dissolved.
3. The standard curve of absorbance vs concentration of a starch/ iodine is prepared.
4. The solution of substrate concentration effect and temperature effect also prepared.
Temperature effect
The equation of standard curve
Y=0.6823x-1.3111
Y= absorbance
x= the concentration of starch
The equation of standard curve
Y=0.6823x-1.3111
Y= absorbance
x= the concentration of starch
Temperature: 40 degree Celcius
The equation of standard curve
Y=0.6823x-1.3111
Y= absorbance
x= the concentration of starch
The equation of standard curve
Y=0.6823x-1.3111
Y= absorbance
x= the concentration of starch
DISCUSSION
Based on result the substrate concentration effect, the rate of reaction enzyme increased due to increasing concentration of substrate. This is because there will be more active molecule for a reaction occur. More active molecules give more chemical reactions since there will be more successful collisions at the active site of the enzyme and substrate. The rate of reaction is directly proportional to the enzyme concentration. This is because the enzyme concentration is increased which enables active sites to react with substrates, increase the rate of reaction.
In this solution of starch and amylase there are only a few amylase molecules to collide with the starch substrate. This means is less chance of collision occurring and a less probability of the enzyme being 'locked' by the substrate starch 'key'. This means there will be less effective collisions, reducing the rate of reaction.
For temperature effect, the increasing of temperature will increases the Kinetic energy of the molecules. Amylase molecules will therefore collide more, resulting the starch to be taken up by the active sites of the amylase, broken down quicker and maltose to be released. This will cause an increase in rate of reaction. However after the optimum temperature, which is above 40 c, the ionic and hydrogen bonds holding the amylase together will brake and denature, so the reaction will no longer occur. I will keep the temperature constant by keeping it at a mild temperature so the enzymes do not denature.
Based on result the substrate concentration effect, the rate of reaction enzyme increased due to increasing concentration of substrate. This is because there will be more active molecule for a reaction occur. More active molecules give more chemical reactions since there will be more successful collisions at the active site of the enzyme and substrate. The rate of reaction is directly proportional to the enzyme concentration. This is because the enzyme concentration is increased which enables active sites to react with substrates, increase the rate of reaction.
In this solution of starch and amylase there are only a few amylase molecules to collide with the starch substrate. This means is less chance of collision occurring and a less probability of the enzyme being 'locked' by the substrate starch 'key'. This means there will be less effective collisions, reducing the rate of reaction.
For temperature effect, the increasing of temperature will increases the Kinetic energy of the molecules. Amylase molecules will therefore collide more, resulting the starch to be taken up by the active sites of the amylase, broken down quicker and maltose to be released. This will cause an increase in rate of reaction. However after the optimum temperature, which is above 40 c, the ionic and hydrogen bonds holding the amylase together will brake and denature, so the reaction will no longer occur. I will keep the temperature constant by keeping it at a mild temperature so the enzymes do not denature.
Conclusion
The results of the experiment showed that lowering the concentration of enzyme and adding more water, the absorbance of the solution decreases. Therefore, our experiment supports our hypothesis that higher concentration of enzyme will increase the rate of reaction. Higher concentration of enzyme - higher absorbance and color intensity.