Enzyme Catalyzed Reaction Lab Report

However, the rate of reaction only increases for a certain period of time until there is lesser substrate molecules than the enzyme molecules. The increase of enzyme concentration does not have effect if there are lesser substrate molecules than enzyme molecules initially.

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

The products produced by the first reaction were used as a substrate for the second. In this case the enzyme used NADH, which resulted in the decreased absorbance due to the NADH oxidation to NAD+. In addition, the spectrophotometer was used as a measuring device to follow the change in absorbance of the NADH molecules at 340nm.

Hypothesis: If the concentration of the substrate is increased, then the rate of enzyme activity will decrease. This is because as the concentration of the substrate increases, there is an increasing amount of occupied active sites at any given moment. This will cause a decrease in the rate of enzyme activity as substrate-active site collisions are increasingly slowed down thus bringing down the rate of enzyme activity.

Substrate concentration also affects the rate of reaction as the greater the substrate concentration the faster the rate of reaction and all the active sites are filled. At this point the rate of reaction can only be increased if you add more enzymes in to make more active sites available.

An enzyme stock was created by crushing a Lactaid pull with a mortar and pestle and dissolving it in 10ml of 0.1M Phosphate buffer. After letting it dissolve in a beaker for a couple of minutes, the enzyme solution of Lactase isolated from Lactaid was filtered into another beaker with a small paper towel. Following filtration, 0.5ml of the stock enzyme solution was added to the 4.5ml buffer, transferring 0.5ml from one tube to the next. This serial dilution took place to determine the optimal dilution that will be used for the experiment. Phosphate buffer was used as the blank in the spectrophotometer which was set at a wavelength of 420.

reaction rate increases. If the temperature of an enzyme gets to high the reaction rate will slow

A spectrophotometer’s purpose is to use colors of the light spectrum to determine the concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission was to determine the protein concentration and the standard curve of the unknown sample of BSA. This, by preparing five dilutions of the unknown solution of BSA together with other known concentrations, and then experimenting by observing how the concentrations were passed through the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this

Enzyme catalysis is dependant upon factors such as concentration of enzyme and substrate, temperature and pH. These factors determine the rate of reaction, and an increase in temperature or pH above the optimum will

What does the changes in the amount of substrate on an enzyme’s reaction effect on?

The hypothesis for substrate concentration of the experiment is that enzyme concentration rate of the enzymatic reaction increases with increasing concentration of the substrate. (Kimball)

Using the yellow tube, which included everything but starch, as the blank, each group zeroed their spectrophotometer. This was done so that any absorbance observed depends only on the amount of starch present, not on any other reagents (buffer, IKI). To zero the spectrophotometer, the wavelength was first set at 580nm, using knob 3 (45). Next, the groups made sure that the light next to “transmittance” was lit, and the chamber to be tightly closed. Having the chamber empty & closed tightly provides reference for the darkest condition possible. Using knob 1, the transmittance was turned until it read 0.0 (45). Before the groups used their blank test tube to zero the spectrophotometer, each needed to wipe the tube with kimwipes to ensure a clean reading. Turning knob 2, each group was then instructed to zero the absorbance, 0.000. Upon removing the blank, each trial was inserted into the chamber (46). The

As an enzyme-catalyzed reaction may be the main reason for a reaction to occur faster, many factors can

Enzymes are biological catalysts, which means it decreases activation energy in reactions. The lower activation energy in a reaction, the faster the reaction rate. Many enzymes alter their shape when they bind to the activation site. This is called induced fit, meaning for the enzyme to work to its full potential it has to change shape to binding substrate. The location of enzyme’s activation site is on the surface of the enzyme, where the binding of substrates take place. Enzyme activity can be influenced by a variety of environmental factors. If the concentration of enzyme is low, and there is a great deal of substrate, then increasing enzyme concentration results in more molecules available to convert substrates to products. Thus, increasing enzyme concentration can increase reaction rate. If substrate concentrations are low, and many of the existing enzymes are idle because of a lack of substrate, then adding enzyme will have no effect on reaction rate. Enzyme concentration affects the enzyme activity, because the more enzyme concentration the faster the reaction rate, until it hits it’s limiting factor. When substrate concentration is increased, it also increases rate of reaction. Temperature plays an important