Proteins are the basic building blocks of cellular structure. They are complex organic molecules with varied structural features. These form a very important molecule, without which life cannot be sustained. The name protein is believed to have been derived from the Greek term Proteios which means ‘primary’ or ‘holding first place’. (Chatterjea, 1993) Berzelius J. J coined this term because he thought that these nitrogenous macromolecules are the most important of biological substances. The importance of proteins is evident from the fact that these molecules play a multitude of roles in maintaining the physiological state in the organism.
They are the main structural components of the cytoskeleton. The natural catalysts in the body known as enzymes are proteins. They form different structural as well as functional molecules like immunoglobulins, hormones, actin and myosin. The broad groups of proteins also include cellular receptors, transport proteins like hemoglobin and storage proteins like ferritin. Enzymes are essential for the survival of all organisms. Without them life would not be possible. They are biocatalysts regulating many physiological functions in the body.
Their action includes the digestion of food materials, absorption of nutrients from the gastrointestinal tract, utilization of nutrients in the peripheral tissues, metabolism of carbohydrates, proteins and lipids, detoxification of toxic products, elimination of these toxic products and maintenance of homeostasis. At least one enzyme will be present in all physiological functions. All these physiological activities permit the normal maintenance of internal milieu and help to maintain health. In pathological states, this homeostasis will be lost and this can lead to alterations in the level of enzymes.
Enzymes are complex protein structures made of many amino acids linked by peptide bonds. Proteins are complex molecules made up of many amino acids linked by peptide bonds. There are 20 amino acids in different sequences and numbers. These molecules invariably contain a carboxyl group and an amino group. Protein structure is normally described at four levels of organization. They are generally called primary, secondary, tertiary and quarternary structures. Primary structure is the linear sequence of amino acids held together by peptide bonds in its peptide chain and is concerned with the function of the protein.
The secondary structure is the three dimensional folding and coiling induced by hydrogen bonds. The hydrogen bonds are low energy non-covalent bonds formed between hydrogen and oxygen atoms. Tertiary structure is formed by steric relationship between amino acids brought closer by folding. This results in the formation of supercoils that is stabilized by side chain interactions like hydrophobic interactions, Vaander Waal forces and disulfide bonds. When a protein consists of two or more peptide chains held together by non-covalent interactions or by covalent cross-linkages, it is referred to as quarternary structure.
Conformation of the protein structure is extremely sensitive to agents like temperature, ultraviolet light and pressure. Temperature has an important role in the regulation of the rate of enzyme reaction. At very high temperatures, proteins including enzymes get denatured and thereby lose their functional ability. Catalase is the heme containing enzyme that helps in detoxifying hydrogen peroxide, a toxic substance generated in the body as a by-product of some metabolic reactions. Hydrogen peroxide can oxidize the cell membrane proteins and cause cellular injury.
This is avoided by the action of catalase enzyme, which converts hydrogen peroxide to water and oxygen. The absence of catalase or a slow action of catalase can result in injury to hepatic cells. Purpose The action of catalase on hydrogen peroxide is affected by many factors like temperature, pH, presence of metallic salts and the concentration of catalase and hydrogen peroxide. The main purpose of the lab was to observe and understand how each of these factors control the rate of catalase reaction. The objective was to learn how extraneous factors affect enzyme reactions.
Hypothesis Influence of temperature To a certain extent, increase in temperature results in an increase in the activity of catalase. (Fraser & Kaplan, 1955) Beyond these limits, if the temperature goes above or below the specified limits, it will affect the rate of conversion of hydrogen peroxide. Influence of pH The role of pH on the rate of catalase reaction is similar to the influence of temperature. There is a certain range of pH at which pH will accelerate the rate of reaction. If the pH goes above that range or below that, the speed of catalase reaction will decrease.
At extremes of pH, the structure of catalase will be altered. Usually the optimal range of catalase activity will be between pH 3. 5 and pH 5. 5. (Chance, 1952) Influence of catalase concentration An increase in the catalase concentration increases the rate of conversion of hydrogen peroxide to water and oxygen. (Caldarera et al. , 1972) This is due to the fact that more catalase molecules are available for acting on hydrogen peroxide. If the catalase concentration is decreased, the reaction will be decelerated. Influence of hydrogen peroxide concentration
Rate of catalase reaction will be decreased by an increase in the concentration of catalase. (George, 1949) When the number of free hydrogen peroxide molecule increases, it requires a corresponding increase in the number of catalase molecules for the reaction to happen at the earlier pace. This shows that there is equilibrium between the concentration of substrate and the concentration of products. A shift in the equilibrium will affect the kinetics of the reaction. Influence of metallic salts Metallic salts cause denaturation of catalase and results in the deceleration of the rate of reaction. Lanyi & Stevenson, 1969) The metallic ions may cause structural changes in the enzyme molecule and affect the reaction. There are other metal activated enzymes also, where the metallic ions activate the enzyme mediated reaction. Sources of errors We had used buffers to dilute hydrogen peroxide and to make solutions at different pH. This could be a source of error as these buffers might affect the reaction of catalase by altering the pH. They could maintain the pH constantly for a certain extent and this might have affected the observations. Another source of error was the overlapping discs which were used for catalase.
This could have caused errors in judging the results and changed the results. Strengths and weaknesses The greatest strength of this lab is that we got a good understanding of enzyme kinetics and the factors affecting it. We could do the procedure correctly so that the source of errors was minimized. Also we increased the number of tries to increase the accuracy of results. The only weakness we found was the insufficiency of time. We couldn’t get enough time to complete the whole reactions most of the times. This was mainly due to the limitation of time provided to execute the whole experiment. Conclusion
We learned the effects of temperature, pH, concentration of enzyme, concentration of substrate and effect of metallic salts on catalase activity. They alter the rate of reaction of catalase on hydrogen peroxide. The rate of reaction varies from one agent to another. Temperature and pH affects the rate of reaction beyond a certain range, within which the reaction was accelerated. A similar accelerated response was seen by increasing the concentration of catalase enzyme. But there was a decrease in the rate of reaction by increasing the concentration of hydrogen peroxide and by adding metallic substrates.