Respective Properties of Macromolecules and Enzymes - Research Paper Example

This study was carried out to identify the different types of macromolecules and to learn about its respective properties. Since no singlestudy can lead to identification of carbohydrates, two tests 1) Benedicts reagent was done to identify monosaccharide, glucose and ribose; 2) Lugol`s iodine was used for the identification of starch, a polysaccharide. Visual observation of simply adding few drops of salad oil in water shows that oil is less dense than water and is immiscible. Enzyme reaction and specificity was studied using the enzymes Tyrosinase and Sucrase. Effect of different physical parameters including temperature, enzyme concentration and pH was determined for the enzyme tyrosinase. Phenylthiourea, an inhibitor was also used to study its effect on the eznyme. Introduction: Every living thing on earth is made of carbon, and each compound containing carbon is known as organic compound. Four kinds of major organic compounds are known carbohydrates, lipids, proteins and nucleic acids. Proteins are organic compounds made up of amino acids joined by peptide bonds between the amino and the carboxyl group of the adjacent amino acids. The proteins can be classified according to their structure; the linear chain of amino acid is called the primary amino acid. In secondary structure there occurs a regular repeating structure that is stabilized by hydrogen bonds. In tertiary structure the stabilization occurs by salt bridges, disulfide bonds, hydrogen bonds. Protein subunits combine together to form the quaternary structure. Enzymes are also an organic compound, basically a protein. “All enzymes are proteins, but all proteins are enzymes”. Enzymes are biocatalyst that speed up the rate of the reaction but do not take part in the reaction. Enzymes have active site, basically a group of amino acids that binds with the substrate, which further carry out the reaction to give a specific product. Enzymes are known for their specificity for substrates, although not all enzymes are specific. The rate of the reaction governed by enzymes is increased when the optimal conditions like temperature, pH, and substrates concentration are provided for the reaction. Two theories are known for the action of enzymes on substrates. 1. The lock and Key Mechanism and, 2) Induced fit theory. Enzymes are also inhibited by inhibitors, which can be substrate analog or any other compound that binds to the enzyme such a manner that the enzymes is unable to function. Inhibitors can be of two kinds, competitive, uncompetitive and non-competitive. (1) Carbohydrates are simple organic compounds, which can be either aldehyde or ketones. They are most common macromolecule and can be found everywhere in plants (in cell wall), in animals and in humans. They are the basic energy providers such as glucose, sucrose, galactose and fructose. They are basically classified into two monosaccharide and polysaccharides. Monosaccharides are the simplest carbohydrates and they cannot be broken down into another compound, whereas disaccharides can be broken down further into two molecules of monosaccharide. A simple example; glucose is a monosaccharide whereas; maltose is a disaccharide (glucose+glucose). Oligosaccharides and Polysaccharides are also carbohydrates; the former is long chain of monosaccharide linked by glycosidic bonds, whereas polysaccharides is also composed of long chains of monosaccharide, the former contains only 2-9 units whereas the latter is composes of more than 9. Starch is also polysaccharide. (1) Lipids are organic molecules made up of fatty acids and glycerol. The main function of lipids is that they are vital source of storage. For example in humans, lipids are in adipose tissues. Lipids are mainly hydrophobic and also amphiphatic; possess both polar and non polar groups. It is immiscible in water. (1) Methods: Colored reactions were observed by using spectrophotometer. For instance, when we added lugols iodine in starch sample, it resulted in brown colour; the intensity of the colour developed was studied using the spectrophotometer. The spectrophotometer was allowed to stabilize for an hour and the instrument were set at the required wavelength. In a cuvette, 1ml of the sample was added and then the cuvette was inserted in the cuvette holder and the respective reading was noted. Results: 1. Benedicts Tests for Monosaccharides: Addition of Benedicts reagent (2ml) into the Tube 1 (glucose sample) followed by heating in boiling water bath for 5 mins leads to orange red colored product, confirming the Glucose is a monosaccharide. Whereas addition of benedicts reagent in tube 2 and 3 (sucrose and starch respectively) did not change its color. The sample remained blue showing that the samples are not monosaccharides. In tube 4 which contain Ribose, addition of Benedict’s reagent changed the color to brown red, thus confirming that the sample is monosaccharide. In tube 4 containing distilled water as sample, no change in color was obtained. The sample after adding reagent remained blue in color. 2. Lugols Test for Starch: Addition of 2 ml Lugols iodine to 2 ml, 1% glucose resulted in brown color. No change in color was observed whereas adding 2ml iodine to 2 ml, 1% Starch led to dark blue colour, clearly indicating that the sample, starch is a polysaccharide. Addition of lugol`s iodine to distilled water did not change its color. The color of the sample remained brown confirming the absence of polysaccharide. 3. Visual tests for lipids: Adding 2-3 drops of salad oil into 10 ml of water resulted into separation of oil from water, after letting the mixture stand for few minutes. This result clearly indicates that oil is less dense than water. 4. Enzymes 4.1 Characteristic of Enzyme reaction: In tube 1, containing enzyme + substrate (Pyrocatechol) after 5 mins resulted in yellow color. The corresponding absorbance at 400nm was 0.690. Tube 2 and 3 containing Pyrocatechol and enzyme Sucrase as controls for 5 mins absorbance at 400nm were 0.016 and 0.002 respectively. 4.2 Effects of Temperature on Enzyme specificity: Enzyme substrate mixture tubes were incubated at different temperatures. At 0⁰C the color developed was light yellow; the absorbance 400nm of this sample after 10 mins was 0.412. At 20⁰C, and 60⁰C colors obtained were mid-yellow. The corresponding absorbance at 400 nm was 0.456 and 0.498. Interestingly, at 60⁰C the color increases from light yellow to dark yellow. The absorbance also increased drastically to 0.698. At 100⁰C, the color obtained was pink yellow and the absorbance was 0.438. 4.3 Effect of enzyme concentration : Tube 1 containing enzyme + DI water+ Pyrocathehol) displayed light yellow in color, these absorbance of which at 400nm is 0.131. Tube 2 and 3 contain enzyme + pyrocathechol + water + enzyme) pyrocathecol showed mid and dark yellow in color respectively. The absorbance at 400nm in tube 2 was 0.414 and tube 3 was 0.905. 4.4 Effect of pH : The pH3 of enzyme substrate mixture in tube 1 results in abs 400 nm, 0.013. The absorbance of samples in tubes 2 and 3 having pH 5 and 7 was 0.226 and 0.910 respectively. The tubes 4 and 5 abs(400nm) were 0.132 and 0.110 respectively. 4.5 Effect of inhibitors: The tube 1 and 2 containing 1 drop of phenylthiourea + 10 drop DI water and 10 drop phenylthiourea abs 10 drop of DI water were clear. The absorbance obtained at 400nm was 0.02 and 0.003 respectively. Only 20 drops of phenylthourea in tube 4was clear and displayed an absorbace of 0.003. Whereas 1 drop of tyrosine and 10 drop of tyrosine+10drop of DI water in tube 4 and 5 showed abs(400nm) of 0.41. Twenty drop of tyrosine and 20 drop of DI water were light yellow in color and displayed abs (4oonm) of 0.39 and 0.410 respectively. Discussions 1. Benedicts Tests for Monosaccharide: Benedict’s reagent is made up of Sodium carbonate, sodium citrate and Cupper (Cu+2) Sulphate. In presence of reducing agent Cu+2 converted to Cu+1 as red precipitate. Being monosaccharide glucose has free aldehyde group which act as reducing agent while in case of starch (polysaccharides)and sucrose (disaccharide) doesn’t have free aldehyde group and that’s why , tubes containing starch and sucrose remains blue while glucose and ribose containing tubes shoes red color after heating. The test clearly indicates presence of monosaccharide or reducing sugar in given sample. 2. Lugols Test for Starch: Lugols reagent consist of Iodine which reacts with starch and gives bluish color while its remain unchanged in case of mono or disaccharide like glucose and sucrose. Here Addison of Lugols reagent in to starch containing tubes give rise to blue color which clearly indicates that starch is polysaccharide while in case of glucose there was no color change indicates presence of mono or disaccharide. 3. Visual tests for lipids: Oil and water dose not mix with each other due to oil is highly hydrophobic in nature. Due to difference in density one can segregate oil from water where oil remains floated on top of the water. The given sample was tested for oil and found to be oil as sample was separated and remains floated over the water layer. 4. Enzymes: Enzymes are biological catalyst and acted upon its substrate to convert it to product. Here enzyme tyrosinase was taken as model system to investigate different aspect of enzymatic processes. In first experiment colorless substrate Pyrocatechol was taken which converted to yellow color compound by action of tyrosinase. There was distinct yellow color development in excremental tube having substrate +enzyme while in case of control tube there was no color development. It was also seen that even though control having enzyme sucrase and substrate Pyrocatechol there was no color development indicates specificity of enzyme. To evaluate effect of various physical parameters on enzyme activity different experiments were carried out by altering single parameters. In first experiments effect of Temperature on enzyme activity was monitored and it was shown that there was increase in enzymatic activity with temperature up to certain level and than again its start decreasing. The reason behind it is Increase in temperature leads to activation of enzymes but simultaneously there was denaturation of enzyme or portion of enzymes start denaturing. After certain temperature the rate of denaturation exceeds rate of activation and hence decrease in overall activity. Similarly it was shown that increase in enzyme concentration leads to increase in product formation. In third experiment effect of pH was determined and it was shown that enzyme has particular pH at which it works optimum, both side of optimum pH there is gradual decrease in activity. Inhibitors are group of compounds which interferes in enzymatic reaction and decreases its activity. We performed experiments to determined influence of inhibitor on enzyme activity and partially characterized the mode of inhibition. Phenylthiourea was used to determine its inhibition and was found to be highly effective, there was no measurable activity was observed in tube containing phenylthiourea. Similarly in one reaction system tyrosine was used along with Pyrocatechol, It was demonstrated that Tyrosine competitively inhibits conversion of Pyrocatechol to colored compound, which clearly indicates both tyrosine and Pyrocatechol binds to active site. References: 1. David Nelson, Michael Cox. “Lehninger Principles of Biochemistry”, 4th edition. W.H.Freeman company Read More