Experiment #5 - Extraction and Analysis of an Enzyme from Wheat

Introduction

Background Information

Nearly all of life's biological processes are carried out by enzymes, proteins that act as biological catalyst in a reaction. Each enzymes reacts with a very specific substance termed the substrate.. During the reaction the enzyme binds to the substrate inducing biochemical changes that transform the substrate into a product. The enzyme itself is not degraded during the process and is therefore free to interact with more of the substrate once the reaction has finished.

In order to measure the activity of an enzyme one can measure the rate of appearance of the product from the reaction. Initially the appearance of product follows a linear increase. However, after some time the rate of its appearance will steadily begin to level off until it reaches a plateau. The reason for this lies in the saturation kinetics of the enzyme-substrate complex. Even with a large amount of substrate there will only be a limited amount of enzyme to react with, thus limiting the total appearance of product.

The velocity of a reaction can be measured by the following equation:

Using a standard curve generated from a set of velocity reactions one can determine the amount of enzyme in an unknown sample by plotting its vo against the curve.

Factors such as temperature and pH can have a significant affect on both the velocity of the reaction as well as the enzyme itself. At higher temperatures enzymatic reactions speed up to a point known as the temperature optimum. However, above that point the enzyme becomes thermally denatured. Likewise most enzymes have a specific pH range that they can function effectively in. If the environment becomes either too acidic or too alkaline the enzymes will be denatured.

In the wheat kernel the three main parts are the 1) the embryo or germ, 2) the starchy endosperm, and 3) the protective covering. The germ portion of the kernel contains the enzyme acid phosphatase which is used by the embryo to break down the starchy endosperm as a source of food. In order to extract this enzyme the cell must be exposed to a detergent such as NP-40 which is used to dissolve the protective membranes and lyse the cell. Once the enzyme has been extracted its velocity of the reaction can be measured against that of purified acid phosphatase made up in the lab. Using Nitrophenol as a substrate (which when broken down turns yellow in alkaline solutions) the velocity of reaction can be effectively measured.

Purpose

The purpose of this lab is to prepare an enzyme extract from wheat germ, and then to determine the amount of acid phosphatase in the extract.

Hypothesis

The lab purified acid phosphatase will show a greater degree of enzymatic activity then will the naturally extracted wheat germ acid phosphatase.

Procedure

For the enzyme extraction 2 grams of wheat germ and 20 ml of enzyme extraction buffer were placed into a mortar and ground up into a homogenous suspension. The suspension was then filtered through two layers of cheese cloth.

For the enzyme assay a reaction plate was obtained, wells labeled, and filled with 1 ml of KOH. Next two 25 ml glass tubes were obtained, labeled, and filled with 10 ml of phosphatase substrate solution. One ml of each tube was pipetted into the appropriate well on the reaction plate to serve as the time 0 values.. Next 0.1 ml of pure acid phosphatase was placed into tube A and 0.4 ml of wheat germ extract was placed into tube B after which both tube were gently shaken to mix the solutions.

1 ml samples of the solutions from both tubes were removed according to the time chart below and placed into the appropriate wells on the reaction plate.

The KOH in the wells stopped the enzymatic reaction as well as reacted with the nitrophenol product to form a yellow precipitate

For measurement of product during the reaction, wells were labeled on the reaction plate and 2 ml of each nitrophenol standard were added. For a visual determination yellow precipitates from wells A1 - A6 and B1 - B6 were visually measured against that of the standards in wells C1 - C6. Results were recorded and a standard curve was generated after which initial velocities were calculated.

Results

TABLE 1 - Reaction Plate A

TABLE 2 - Reaction Plate B

Graphs

Calculations

Initial Velocities

Discussion

In analyzing reaction plates A (Graph A) the results supported the initial hypothesis in that the purified acid phosphatase had a higher yield of enzymatic activity. For both tubes A and B enzymatic activity initially showed a rapid increase followed by a period of non-increasing enzymatic activity and ending with another rapid increase in enzymatic activity. Reasons for the period of non-increasing enzymatic activity may include experimental error in transferring the enzyme-substrate complex out of the tubes and into its corresponding well in a timely manner. The solution may have been transferred out of the tube either too soon or two late to see a visible difference in the level of enzymatic activity. The fact that there is an increase in enzymatic activity in the last samples collected would seem to rule out the possibility that there may have been something wrong with either the purified acid phosphatase or the extracted wheat germ.

In analyzing reaction plates B (Graph B) the results partially support the initial hypothesis in that tube A showed a higher level of enzymatic activity from t=2.5 min. through t=10 min., but after which there was no visible difference in the level of enzymatic activity in comparison to tube B. The pattern of enzymatic activity for tube A was similar to that of the results from reaction plate A and may thus be attributed to similar reasons. However, the enzymatic activity pattern from tube B showed a more progressives increase all throughout the experiment.

One thing to consider in analyzing the results from both reaction plates is that the amount of enzymatic activity was measured visually using a set of standards for comparison. This type of method can be very subjective is therefore subject to error.

In conclusion, this experiment showed that the enzyme acid phosphatase was effective in reacting with the nitrophenol substrate as evidenced by the appearance of the yellow precipitate nitrophenol in an alkaline solution of KOH. The use of a spectrophotometer would yield more accurate measurements as to the level of enzymatic activity (by way of measuring the yellow precipitate) and is thus highly recommended for more conclusive results.

Study Questions

There are 2mg of acid phosphatase in 400ml of the wheat germ extract in tube B.

2.