Spectroscopy was used to determine the formation of product in the experimental tubes. Introduction:
The objective of this experiment was to determine the optimal pH and substrate saturation of alkaline phosphatase. The experiment was divided into two exercises, exercises A and B. the function of this division was to see enzymes working in different conditions. Part A displayed how much enzyme to use and demonstrated works under "normal" conditions. Part B altered some environmental parameters in order to see how that affected enzyme activity. Blanks were also used in the spectrometer. There were many reasons for having blanks and controls. Firstly the goal of this exercise was to measure the formation of product in the experimental tubes with the use of a spectrophotometer. However, several other reagents in the tubes were present because they were necessary for the reaction to occur (such as the buffer, the substrate, and the enzyme itself) that were not the chemicals needing to be measured. Changes in the absorbance due to the substrate being changed into product was what needed to be measured. Therefore, a method was needed in order to exclude these other chemicals from the spectrophotometer reading in order to have obtained clearer results. The blank was the method chosen to achieve the desired reading. The blanks in this experiment included all of the reagents except the enzyme. The blank often serves another purpose in experiments like this one. Sometimes there may be a color change in the experimental tubes due to oxidation of the substrate or other non-enzymatic factors. Since the blanks also contain the substrate, any oxidation will occur in the blanks as well. Therefore when the spec 20 is "zeroed" with the blank before measuring the absorbance of the experimental tube, any absorption due to oxidation would not show up in the spectrophotometer reading of the experimental tube. Enzyme concentration and substrate concentration also play a role in enzymatic activity. The more enzymes available, the quicker the reaction will occur until the substrate is all used up. More substrates will also mean quicker activity, until the enzyme is fully saturated so that it cannot continue increasing its activity. Activators and inhibitors interact with an enzyme so that its activity is altered. If a molecule increases the rate of reaction, it is an activator, whereas an inhibitor decreases or stops the activity. These substances regulate how fast an enzyme acts. Inhibitors work by unfolding or destabilizing bonds, thus denaturing the enzyme. Some inhibitors block or change the shape of the active site. The pH of an enzyme’s environment measures the acidity, or hydrogen ion concentration, in a solution. If the pH is lowered, the side chains will begin picking up hydrogen ions from its environment, which might disrupt its function. Likewise, as pH is raised, enzymes will lose hydrogen ions and lose its active shape. Hence, maintaining a neutral pH is necessary for most enzymes. Of course, there are also exceptions with stomach enzymes, such as pepsin (pH 2). Experimental section:
20 Test Tubes
Cuvette rack Test tube rack
The Spectrophotometer was set to wavelength 410nm.
A blank test tube was made first by Placing the buffer in the tubes first. Then adding deionized water and then the enzyme. 3.
5 cuvettes were labeled 1E, 2E, 3E, 4E, 5E. The required reagents were added to the tubes. 4.
Using the correct blank, set the range of the Spec 20 using the procedure you learned in the 5.
Spectrophotometry Lab Exercise. Remember: before each measurement of an experimental tube 6.
The spec 20 with the specific blank paired to that experimental tube was "zeroed". Using the procedure learned from the Spectrophotometry Lab Exercise, measurements were taken immediately and continued every 30 seconds for 4.5 minutes. For exercise B:...
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