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Determination of Enzymatic Activity in Glucose Using Dinitrosalicylic Colorimetric Method and Effect of pH and Temperature on Invertase Activity Silva,

J., Subijano, M., Sunglao, A., Supan, E., Tan, C., Tayag, P. Group # 7, 2G Medical Technology, Faculty of Pharmacy, UST

Abstract An enzyme is a protein-based substance which serves as a catalyst in living organisms by regulating the rate of spontaneously chemical reactions. In this experiment, dinitrosalicylic (DNS) colorimetric method was used in the assay of glucose to monitor its enzymatic activity and effect of temperature and pH on invertase activity was determined. Results showed that 3,5-dinitrosalicylic acid was converted into 3-amino-5-nitrosalicylic acid, which contributed to the change of the yellow mixture into red-brown color and in the process, glucose was also converted into gluconic acid. Invertase may be used over an extended temperature an optimum of 50C. The data gathered was plotted into graphs and showed the linear trend of the standard curve having the best fit line for the glucose assay, U-shaped curve for the effect of pH and imperfect bell-shaped curve for the effect of temperature.

Introduction The objectives of the experiment were: (1) to monitor the enzymatic activity of glucose using dinitrosalicylic colorimetric method; (2) to explain the mechanism involved in the glucose assay; (3) to extract invertase from Bakers yeast and; (4) to determine the effects of changes in pH and temperature on reaction rates of an enzyme-catalyzed reaction and; (5) to generate curves that represent the data gathered. Of all the functions of proteins, catalysis is probably the most important. In the absence of catalysis, most reactions in biological systems would take place far too slowly to provide products at an adequate pace for a metabolizing organism. The catalysts that serve this function in organisms are called enzymes. [1] Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. All known enzymes are proteins. They are high molecular weight

compounds made up principally of chains of amino acids linked together by peptide bonds. Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000. [2] Invertase or sucrase is an enzyme that helps in the hydrolysis of sucrose into glucose and fructose. The official name for invertase is beta-fructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal nonreducing beta-fructofuranoside residues in beta-fructofuranosides. Note that alpha-Dglucosidase, which splits off a terminal glucose unit, can also catalyze this reaction. Note that sucrose can be hydrolyzed relatively easily; the reaction proceeds in an acidic environment without the aid of invertase. Invertase is mainly used in the food (confectionery) industry where fructose is preferred over sucrose because it is sweeter and does not crystallize as easily. However, the use of invertase is rather limited

because another enzyme, glucose isomerase, can be used to convert glucose to fructose more inexpensively. For health and taste reasons, its use in food industry requires that invertase be highly purified. A wide range of microorganisms produce invertase and can, thus, utilize sucrose as a nutrient. Commercially, invertase is biosynthesized chiefly by yeast strains of Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Even within the same yeast culture, invertase exists in more than one form. For example, the intracellular invertase has a molecular weight of 135,000 Daltons, whereas the extracellular variety has a molecular weight of 270,000 Daltons. In contrary to most other enzymes, invertase exhibits relatively high activity over a broad range of pH (3.5--5.5), with the optimum near pH = 4.5. The enzyme activity reaches a maximum at about 55C. The Michaelis-Menten values of various enzymes vary widely, but for most enzymes Km is between 2 mM and 5 mM. The MichaelisMenten value for the free enzyme is typically approx. 30 mM. [3] Every enzyme has a temperature range for optimum activity. Outside the temperature range, the enzyme is rendered inactive and is said to be totally inhibited. This occurs because as the temperature changes, this supplies enough energy to break some of the intramolecular attractions between polar groups (Hydrogen bonding, dipole-dipole attractions) as well as the hydrophobic forces between non-polar groups within the protein structure. When these forces are disturbed and changed, this causes a change in the secondary and tertiary levels of protein structure, and the active site is altered in its conformation beyond its ability to accommodate the substrate molecules it was intended to catalyze. Most enzymes (and there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value which varies according to each individual. This can happen if body temperature gets too low (hypothermia) or too high (hyperthermia). [4]

Figure 1. Changes in Enzyme Activities with the Temperature Changes in the pH or acidity of the environment can take place that would alter or totally inhibit the enzyme from catalyzing a reaction. This change in the pH will affect the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule no longer fit, and the chemical change would be inhibited from taking place as efficiently or not at all. In an acid solution, any basic groups such as the nitrogen groups in the protein would be protonated. If the environment was too basic, the acid groups would be deprotonated. This would alter the electrical attractions between polar groups. Every enzyme has an optimum pH range outside of which the enzyme is inhibited. If the pH drops in the blood called acidosis, then enzymes in the blood will be inhibited outside their optimal pH range. If the pH climbs to an unacceptably high value called alkalosis, then enzymes ceases to function effectively. Normally, these conditions do not take place because of the highly efficient buffers found in the blood that restrict the pH of the blood to a very narrow range. [4]

of carbohydrates. Glucose is also sometimes called dextrose. Corn syrup is primarily glucose. Glucose is one of the primary molecules which serve as energy sources for plants and animals. It is found in the sap of plants, and is found in the human bloodstream where it is referred to as blood sugar. [5]

Figure 2. Changes in Enzyme Activities with the pH Correcting pH or temperature imbalances will usually allow the enzyme to resume its original shape or conformation. Some substances when added to the system will irreversibly break bonds disrupting the primary structure so that the enzyme is inhibited permanently. The enzyme is said to be irreversibly denatured. Many toxic substances will break covalent bonds and cause the unravelling of the protein enzyme. Other toxic substances will precipitate enzymes effectively removing them from the solution thus preventing them from catalyzing the reaction. This is also called denaturation. [4]

Figure 4. Chemical Structure of 3,5Dinitrosalicylic acid Dinitrosalicylic acid (DNS, DNSA, 3,5dinitrosalicylic acid or 3,5-dinitro-2hydroxybenzoic acid) is used in colorimetric determination of reducing sugars and to analyze glycosidase (glycoside hydrolase) activity by quantitation of enzymatically released reducing sugar. [6] Dinitrosalicylic colorimetric method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions: Aldehyde 3,5-DNS group
reduction

oxidation

carboxyl

group acid

3-amino,5-nitrosalicylic

Figure 3. Chemical Structure of Glucose Glucose is a carbohydrate, and is the most important simple sugar in human metabolism. Glucose is called a simple sugar or monosaccharide because it is one of the smallest units which have the characteristics of this class

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The above reaction scheme shows that one mole of sugar will react with one mole of 3,5-

dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicated than that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as decomposition of sugar also competes for the availability of 3,5-dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of the developed color. [7] Materials and Methods A. Materials In this experiment, the materials needed are Bakers yeast, Erlenmeyer flasks, 0.1 M buffer solutions (pH 2, 3, 5, 6, 7.5, 7.5, 8,

11), Paraffin film, sucrose solution, 1% glucose standard solution, Dinitrosalicylic acid (DNS) reagent, pipettes, aspirator, test tubes, beaker, thermometer, hot plate, analytical balance and UV-Vis spectrophotometer. B. Methods 1. Glucose Assay Using Dinitrosalicylic Colorimetric Method A set of test tubes were prepared by following the table below (Table 1). 3 mL of DNS reagent was added in all of the test tubes. After mixing, the test tubes were immersed in the water bath for 10 minutes at 95C to develop the characteristic red-brown color. The test tubes were then cooled to room temperature and the absorbance was measured at 540 nm using a spectrophotometer.

Table 1. Preparation of Standard Solution Test tube # 1 2 3 4 5 6 7 8 Amount of 1% Glucose Solution, mL 0.00 0.30 0.50 0.80 1.20 1.50 1.80 2.10 Amount of Distilled Water, mL 3.00 2.70 2.50 2.20 1.80 1.50 1.20 0.90

2. Extraction of Invertase from Yeast Using an analytical balance, 0.25 g of Bakers yeast was weighed. The yeast was then dissolved in distilled water to make a 250-mL solution. The mixture was allowed to stand for 20 minutes at room temperature. The supernatant was collected when sedimentation occurred and served as the enzyme stock solution. 3. Preparation of Denatured Invertase Stock Solution

One hundred mL of the enzyme stock solution was incubated in a boiling water bath for 10 minutes. After heating, the solution was cooled to room temperature. The supernatant was collected when frothing occurred and served as the denatured enzyme stock solution. 4. Effect of pH on Invertase Activity Eight sets of three test tubes were prepared. In each set, the first test tube was dropped with 0.50 mL of denatured enzyme

stock solution. The second test tube, which served as trial one, and the third test tube, which served as trial two, were placed each with 0.50 mL of the enzyme stock solution. In the first set of test tubes, 1 mL of 0.1 buffer solution with pH 2 was dropped. The same was done with the other seven sets of test tubes, but with varying pH levels of 3, 5, 6, 7.5, 7.5, 8, and 11 of the buffer solutions. 1.50 mL of sucrose solution was placed in all of the test tubes. The test tubes were then incubated in 60C for 15 minutes. After heating, 3 mL of the DNS reagent was added in all of the test tubes. For 10 minutes at 95C, the tubes were again placed in the water bath. The test tubes were cooled to room temperature and the absorbance was measured at 540 nm. 5. Effect of Temperature on Invertase Activity Water baths with varying temperatures of 20, 30, 40, 50, 60, 70, 90 and 95C were set up. Seven sets of three test tubes were prepared. In each set, the first test tube was dropped with 0.50 mL of denatured enzyme stock solution. The second test tube, which served as trial one, and the third test tube, which served as trial two, were placed each with 0.50 mL of the enzyme stock solution. 1 mL of buffer solution at pH 5 and 1.50 mL of sucrose solution were added into the test tubes. Each set of test tubes were placed on each of the water baths with different temperatures and incubated for 15 minutes. After heating, 3 mL of the DNS reagent was added in all of the test tubes. For 10 minutes at 95C, the tubes were again placed in the water bath. The test tubes were cooled to room temperature and the absorbance was measured at 540 nm. Results and Discussion 1. Glucose Assay Using Dinitrosalicylic Colorimetric Method

In the experiment, color change of the solution with dinitrosalicylic acid, a yellow solution, into red-brown solution was observed with the presence of heat. This is due to the conversion of the 3,5dinitrosalicylic acid to 3-amino-5introsalicylic acid, which accounted for the red-brown solution. Dinitrosalicylic acid also reacted to glucose, which was reduced to gluconic acid. Oxidation is a reversible chemical reaction and its reverse reaction is reduction. Since the absorbance at 540 nm is linearly dependent on the concentration or mass of glucose, the reaction can be used for quantification of reducing sugar. In solving for the amount of hydrolyzed glucose (mg/mL), the formula C1V1 = C2V2 was used. Using the formula, the following were obtained: = 0 mg/mL = 0.1 mg/mL = 0.166 mg/mL = 0.266 mg/mL = 0.4 mg/mL = 0.5 mg/mL = 0.6 mg/mL

Table 2 shows the amount of hydrolyzed glucose and the corresponding absorbance, which is the average of the absorbance values for Trial 1 and Trial 2.

Table 2. Amount of hydrolyzed glucose and the corresponding absorbance

Test tube number 1 2 3 4 5 6 7

Amount of hydrolyzed glucose (mg/mL) 0.00 0.1 0.166 0.266 0.4 0.5 0.6

Absorbance 2.018 2.77 2.76 2.83 2.9 2.93 2.78

Figure 5. Absorbance VS. Concentration of Hydrolyzed Glucose found to be y=0.9325x + 2.4419. It also shows a linear trend, which indicates accuracy of the data gathered. 2. Effect of pH on Invertase Activity Invertase can be affected by pH. An extremely low or high pH can result in complete loss of activity for most enzymes.

Figure 5 shows a straight line, the standard curves best fit line and the plotted points which indicate amount of hydrolyzed glucose and the corresponding absorbance. The line was computed through the linear regression function of a scientific calculator and the slope-intercept form calculated was

Table 3. pH and corresponding amount of sucrose hydrolyzed pH 2 3 5 6 7.5 7.5 8 11 Amount of sucrose hydrloyzed (mg/mL) -2.297 -2.588 -2.502 -2.619 -2.556 -2.619 -2.691 -2.119

Figure 6. Amount of sucrose hydrolyzed VS pH

In Figure 6, a U-shaped graph is formed. The result of the graph is very far from the ideal one, which is a bell-shaped graph. The U-shaped curve was due to the negative values of the amount of sucrose hydrolyzed. This kind of error was committed due to some possible factors like the improper preparation of the mixtures or the spectrophotometer itself committed errors.

3. Effect of Temperature on Invertase Activity Temperature affects the invertase activity. A very low temperature will not allow the enzyme to react while a very high temperature will denature the protein.

Table 4. Temperature and corresponding amount of sucrose hydrolyzed Temperature (C) 20 30 50 60 70 90 95 Amount of sucrose hydrolyzed (mg/mL) -2.484 -2.594 -1.986 -2.288 -2.306 -2.361 -2.588

Figure 7. Amount of sucrose hydrolyzed VS. Temperature Date and time retrieved: January 15, 2014, 12:25 AM Figure 7 shows that the optimum temperature was obtained at 50C. The highest peak at the curve represents the highest concentration which was determined by the absorbance. Temperature above 50C shows that invertase reached denaturation. References Book:
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