Enzyme and Protein, Essay Example

Introduction

Invertase is an enzyme derived from yeast which hydrolyzes sucrose into and fructose and glucose. Invertase is officially called beta-fructofuranosidase (EC3.2.1.26), implying that the enzyme catalyzes the hydrolysis of the terminal beta-fructofuranoside which are non- reducing residues in beta-fructofuranosides. Yeast invertase demonstrates basic reaction similar to that of a ß-fructofuranosidases many of which undergo hydrolyzation by the enzyme action; however the activity yeast invertase towards sucrose exceeds that observed with other ß-fructofuranosides which occur naturally like stachyose and raffinose

Invertase is mainly used confectionery industries which prefer fructose over sucrose because it does not crystallize easily like sucrose and is sweeter. For taste and Health reasons, invertase should be highly purified for it to be used in food industry. A large number of microorganisms utilize sucrose as a nutrient because they can produce invertase. Invertase activity has been demonstrated in molds, yeast, plants and higher animals and bacteria. Invertase is commercially biosynthesized mainly by strains of yeast; Saccharomyces carlsbergensis or Saccharomyces cerevisiae.

The hydrolytic reaction of invertase is assayed conveniently by observing the change in reducing capacity and optical rotation when incubated with sucrose the assays are called fixed –time assays and kinetic assays respectively.  This exercise utilizes the fixed-time assay procedure in which alkali is used to stop the reaction and subsequently the quantity of reducing sugar measured by 3, 5-dinitrosalicylate method.

Abstract

This experiment is intended to isolate the enzyme invertase which is involved in hydrolysis of sucrose and purify the enzyme, perform quantitative and qualitative analyses of each fraction collected, and then characterize the catalytic nature of the partially purified enzyme.

In this experiment a protein standard will be developed to be used in the protein assay.  Three fractions of the protein will be developed for use in determining the purification of invertase.  Dried yeast cells will be disrupted by autolysis, cell debris removed, and the total activity and specific activity of the crude autolysate determined.  The crude enzyme will then purified by removing the contaminating materials like proteins and other molecules through precipitation as their picrate salts, followed by acetone fractionation of the remaining preparation. The percent yield, total activity and specific activity will be calculated for each step in the fractionation. After purification the catalytic properties of the enzyme will be studied under different conditions by varying, temperature, pH and adding inhibitors. The aspects of the catalytic behavior of the purified enzyme to be studied will include Vmax, Km, optimum pH, optimum temperature and inhibition characteristics. Lineweaver-Burk plot will be used for displaying the data as well as identification of Michaelis-Menten Michaelis-Menten kinetic data will be collected using two different inhibitors which will be added in the sucrose enzyme mixture and the Vmax and Km values will be obtained from the graphs prepared each time a different inhibitor is added. The lineweaver –Burk plots will also be utilized for identify the type of inhibition demonstrated by the two inhibitors .The enzyme activity will also be determined at different temperatures and PH  to determine the optimum temperature and PH  for the enzyme invertase.

Many of these aspects of enzymes are important in understanding enzyme kinetics. Enzymes are very important molecules in life of every organism for they play vital roles in biochemical pathways necessary for the survival of the organisms.  They speed up these reactions by lowering the activation energy, which must be overcome for a reaction to occur and hence enable the metabolic reactions to keep up with the phase required by the body.   Without enzymes life simply would cease.

Results

Table 1.  Protein Assays

Table 2.  Enzyme Activity Assays

Table 3.  Results of the Purification of ß-Fructofuranosidase From Yeast

Table 4. Kinetic and Inhibitor Assays

Table 5. Temperature Optimum Assay

Table 6. pH Optimum Assay

Discussion

The experiment was intended to determine the concentration of proteins in the sample.  By centrifugation, the protein was separated into three different fractions all of which were had different concentrations.  The concentrations of each fraction sample were read and determined by spectrophotometry. In spectrophotometry, the higher the concentration the higher the absorbance since the many protein molecules absorb light as it passes through the cuvette

The results from various fractions were as follows

Fraction 1  

Dilution                                        Protein Concentration (uMol)

1/10^th                                                                    103

1/100^th,                                                                30

1/1000^th,                                                              6

1/10,000^th                                                           2.5

This showed that the concentration of the proteins reduced with increased solvent volumes. This means that the proteins were less with every increasing dilution. Conversely the absorbance reduced with every subsequent dilution.

Fraction 2

Dilution                                        Protein Concentration (uMol)

1/10^th                                                                    83

1/100^th,                                                                16

1/1000^th,                                                              2

1/10,000^th                                                            0

Fraction 3

Dilution                                        Protein Concentration (uMol)

1/10^th                                                                    97

1/100^th,                                                                28

1/1000^th,                                                              4

1/10,000^th                                                            0

This sample was followed by an enzyme assay to determine the enzyme concentration as well as determine the concentration of glucose and fructose present in the sample. Samples were analyzed by spectrophotometer.

Fraction 1

Dilution                               Absorbance    Protein Concentration (uMol)

1/10^th                                            0.58                        1.83

1/100^th,                                        0.54                         1.74

1/1000^th,                                      0.06                        0.04

1/10,000^th                                      0                             0

Fraction 2

Dilution                               Absorbance    Protein Concentration (uMol)

1/10^th                                            0.54                        1.74

1/100^th,                                        0.39                        1.25

1/1000^th,                                      0.02                      0.113

1/10,000^th                                      0                            0

Fraction 3

Dilution                               Absorbance    Protein Concentration (uMol)

1/10^th                                            3.0                        10.16

1/100^th,                                        2.3                        8.04

1/1000^th,                                    0.64                       2.05

1/10,000^th                                  0.03                      0.12 

Fraction three had very high protein concentrations as well as total protein concentrations as compared to fraction 1 and Fraction 2. This implies that the bulk of the proteins precipitated to the bottom of the tube hence leading to the high concentrations in fraction 3. This is further asserted by the manner in which there is gradual increase in concentration moving from fraction 1 through two then to three.

The velocity ratios also demonstrate the increase in movement through the matrix as a function of concentration. As the concentration increases, the velocity also increases. Similarly increase in inhibitor concentration led to increase in velocity since the inhibitor reduces the ability of the enzyme matrix to bind to the substrate and therefore increasing the speed of elution of the sample.

The results for the kinetic and inhibitor assays are found in table 4 are a Michaelis-Menten plot for sucrose.  The table has three entries, one for the standard, one for the sucrose with low inhibitor and one for sucrose with high inhibitor. The tables demonstrate the effect of the inhibitor on the catalytic ability of the enzymes. The inhibitors function to reduce the catalytic ability of the enzymes since the function to reduce the concentration.

It is important to notice that the Km’s for the low and high inhibitors do not vary from one another by a significant amount.  This is because the inhibitors are not competitive with one another for sucrose. This is known as uncompetitive inhibition.  The Lineweaver-Burk plot it demonstrates that a ternary complex is formed due to the lines crossing neither on the 1/V axis or the 1/[S] axis.

The Lineweaver-Burk plot also demonstrates that each inhibitor has its own Km and its own Vmax.

A temperature optimum assay was also preformed in this laboratory exercise.  When viewing the temperature optimum graph (graph 5) it demonstrates as temperature increases to high the enzymes ability to optimally perform diminishes.  At approximately 70 degrees Centigrade the velocity is zero.  The optimum temperature for the enzyme is approximately 50 degrees Centigrade.  This was based on the graph.   At the other extreme when the temperature is very low the ability of the enzyme to catalyze the reaction is impeded. At 2 degrees Centigrade the velocity is .008 indicating not much reaction is occurring.

A pH optimum assay was also preformed on the enzyme.  The optimum pH at which the enzyme functions seems to be approximately 4-4.1.  When the pH was varied the velocity changed in accord.  When the environment was acidic with a pH of approximately 2.5 the enzyme did not function.  There was some record of velocity however it is very low.  At the pH of 2.5 the velocity is .03.  At the other extreme a pH of approximately 7.4 the velocity is zero indicating the enzyme is not functioning.  It is important to understand that different enzymes have different pHs at which they function optimally. This is all based on protein conformation where the different proteins have their isoelectric points. Outside these points, the proteins get denatured hence they lose their functionality.

Conclusion

This exercise coupled many different aspects concerning enzymes together to develop a through understanding for their isolation.  A protein standard was developed to identify unknown amounts of protein in samples.  A fractionation system was used to purify the protein.  Enzyme activity standards were developed to compare each fraction with.  Michaelis-Menten plots and Lineweaver-Burk plots were used to characterize the enzyme.  Temperature and pH optimum assays were developed to gain further understanding of the enzyme.  Knowledge of all of these techniques made this a unique and strong experiment.

Proteins and entire protein functions are based on conformational and structural integrity. Any denaturing agents tend to destroy this integrity and hence make the proteins lose their function. This is underlined more by the fact that all the denaturing agents namely pH, heat as well as temperature reduce protein activity. Enzymes are also proteins and all denaturing agents have a similar effect on the enzyme as they have on the proteins. Enzyme activity is therefore optimum at a given range of temperature and pH.

Works Cited

Buyer, R.F. “Modern Experimental Biochemistry” Addison-Wesley publishing Co., Inc.

Lehninger, A., Nelson, D., Cox, M. “Principles of Biochemistry” 2^nd edition. Worth Publishers, Inc. 1993;211-222.

Mills, R. Exercise 5 Purification and catalytic properties of yeast invertase. 1998.

Norman-Tiner, C.  Biochemistry Lecture. Summary handout.

Sperelakis, N. ed. “Cell Physiology”. Academic Press 1995;96-103.