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.

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

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.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

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.

In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube

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

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

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

Independent: Substrate Concentration: Throughout the experiment, the concentration of the substrate used will be increased in order to determine the effect of an increase of substrate concentration on enzyme activity. The substrate throughout the experiment will be a hydrogen peroxide solution and an original 30% concentration will be diluted with water into 10% and 5% concentrations in order to observe the effect of

The addition of sodium hydroxide at different times means that the amount of Nitrophenolate anion released will be different, thus, will have different absorbance readings (generally lower). The more time the enzyme is exposed to the substrate without the addition of

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.

The ending result of this experiment confirms that as five test tubes are lined up with the varying level of absorbance, different results in the level of absorbance will appear as well, this is visible in above table. Thus, this is due to the varying amount of water in the solution. The blank sample had a 0.30 in its level of absorbance.

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)