Enzymes - Biological Catalysts

Introduction

        Enzymes are biological catalysts that speed up chemical reactions. Enzymes are important for all living organisms because their biochemical reactions help with digestion and reproduction of gene information (Cell Research). The enzyme reacts with a substrate to form a new compound called the product. Enzymes speed up the rate of reactions by lowering the energy of activation needed to start the reaction. These reactions can vary based on the environmental conditions. A rise or lowering in pH, and temperature levels. Something else that can affect the enzyme from working properly is an inhibitor. An inhibitor interferes with the action of an enzyme (NCI).

        A type of enzyme found in animals, bacteria, fungi, and plants is amylase. Amylase controls the digestion of starch into molecules of maltose. Starch is an important source of energy for cellular respiration. The chemical DNS can be used to measure the amount of maltose in a solution. When DNS is reacted with maltose, it turns red which can be used in a spectrophotometer to measure how much light of a wavelength is absorbed by a solution.  The following experiment was carried to determine a standard curve using the concentration of maltose to the absorbance of a solution.

Materials and Methods:

The experiment in exercise 1 involved generating a standard curve to calculate the amount of maltose in a sample.  The procedure involved using absorbance measurements to identify maltose concentrations in a solution with DNS, which where a standard curve will be created. The curve was created using known concentrations of maltose with DNS to produce an equation relating the concentration of maltose to the absorbance of a solution.

 Table 1

Legend: Table 1 shows that five test tubes were used with 1 mL of 1% starch solution added. As well as the desired maltose percentage for maltose or buffer, and 1 ml of DNS reagent.         

As shown in table 1, 1 mL of starch solution was added to each tube, then the appropriate maltose solutions or buffer. Then 1mL of DNS reagent was added to each tube, and covered with a parafilm square. The tubes were placed in a 95 degree C for five minutes; while that was happening a set of dilution tubes was prepared by adding 4mLs of dH2O. The wavelength of the spectrophotometer was set to 540 nm and a mode of 0-2A. The tubes A1 through A5 were read by the spectrophotometer; the absorbance reading can be seen in Table 2.

Table 2

Legend: Table 2 shows how each of the test tubes were read in the spectrophotometer. A1 was at 0.0 because it was the control, and A2 through A5 the readings increased.         

Based on the experiment shown in table 1 and 2, a hypothesis was created. It stated that based on a high maltose level, the absorbance reading would be higher.

Next involved 5 test tubes labeled B1 through B5, and 1 mL of buffer to B1 was added. 1mL of different concentration of amylase indicated the table to each tube B2 thorough B5.

Table 3

        Similar to experiment 1, the diluted contents of each reaction tube was 1:5 ratio with distilled water before the reading absorbance. The samples were taken to the spectrophotometer. The absorbance measurement was at a wavelength of 540 nm using B1 as the blank.

Chart 1

[pic 1]

Chart 1 shows that there was a correlation on absorbance level and higher maltose percentage. The equation above shows mx+b is the rate of reaction.

        Exercise three was hypothesized that in increased concentration would also increase the rate of reaction. Exercise 3 tested the effect of different concentrations of substrate on the reaction rate. Five test tubes labeled C1 through c% were placed in a tube rack and 1ml of buffer was added to c1, and 1ml of the appropriate concentration of starch shown on the chart below was added to each tube. 1ml of the 2.0mg/ml amylase solution was added to each tube in 5-10 second intervals.

Table 4