Analyzing Protein - Research Paper Example

Analyzing Protein This experiment focused on the effects of enzyme concentration and pH on peroxidase enzyme activity. Peroxidase enzyme was extracted from radishes. This experiment was based on the principle that peroxidase catalyses the reduction of hydrogen peroxides into water by oxidizing organic substrate and guaiacol was used as a substrate. Several different concentrations were assayed to determine the effect of enzyme concentration after which dilutions of 1x, 2x, 4x, 6x, 8x, 10x, and 20x were prepared in spectrophotometer tubes and 1 ml 40mM guaicol added before mixing the content immediately. The absorbance were then read at 10 seconds for 1x and 2x tubes, at 20 seconds intervals for 4x and 6x tubes and 30 second intervals for 8x and 10x tubes. The effect of pH was tested by adding 0.5ml of H2O2 and 0.5 of appropriate buffer to spectrophotometer tubes containing the volume of water and extract that gave an enzyme activity rate of 0.1unit/minute and absorbance read for pH levels; 3, 4, 5, 6, 7, 8, 9 and 10. For Enzyme kinetics, Spectrophotometer was blanked and the contents of 50 microml guaiacol tube added to tubes of diluted enzymes before reading the absorbance levels and calculating the initial velocity of each substrate concentration. The results were then recorded. From the data from the table, Lineweaver-Burk plot of 1/v vs. 1/(S) and used to calculate Vmax and Km of guaiacol. The results shows that peroxidase enzyme is specific to pH 4 while enzyme reaction is directly proportional to concentration optimal concentration ensures that the rate of reaction is high and steady for a long period of time Introduction Enzyme is a molecule of protein present in the living cells and act as catalysts for various biological reactions. Enzymes function in three different categories; they act as catalysts and thus speed up the rate of chemical reactions. As stated by Onteh et al (2005), many reactions takes place in the cell of organisms a million times faster in the presence of enzymes. The second characteristic of enzyme is that enzymes are specific to reactants called substrates. In this respect, enzymes can only act on specific reactants and not all to produce products. The third and most important characteristic of enzyme is that its rate of activity can be regulated from high rate of reaction to low and vice versa. This characteristic property is of particular importance to many chemical reactions taking place within and outside the ling cell. It is also important to note that ability to control enzymes that catalyze a certain reaction taking place within the cells ensures right reactions are allowed to proceed, at the right time and place. Cells can therefore control the production of different enzymes at different times. The reason for high specificity and efficiency of an enzyme is attributed to the nature of the interaction between the enzyme and the substrate commonly known as enzyme-substrate reactions. In this reaction, a complex is formed between the two molecules with non-covalent weak interactions on the active sites thus there is no covalent bond at the active sites. This kind of interaction increases and facilitates the rate of conversion of the substrate into the product. It is also important to note that the substrate molecule binds to only a specific active region of the enzyme-substrate complex (Onzález et al, 2014). The reason for this is that these regions are highly selective to substrate that can bind with the enzyme. The rate of enzyme activity is an important area of study since we will be able to control a reaction catalyst by an enzyme. Just like all catalysts, enzymes are proteins that speed up the rate of a chemical reaction without being used in the process thus a small amount of enzyme can catalyze the conversion of large quantities of substrates into product. This is achieved through lowering of the activation energy required to convert reactants into products (Campbell et al 2008). However, the rate of enzyme activity is affected by many factors that affect protein since enzymes are protein in nature. Particularly, factors that affect protein activity, factors that result into protein denaturation and protein folding will definitely affect enzyme. These factors include; pH, organic solvents, salt concentrations and temperature. pH also affects active sites without necessarily affecting the structure of the enzyme. Temperature can however affect enzyme catalyzed reaction by either by increasing the energy level of the substrate or by changing the protein shape. The amount of enzyme available can also affect the rate of reaction since the more enzymes the higher the rate. Enzyme kinetics refers to the study of the rate at which enzymatic reaction occurs. A part from investigating the factors affecting enzymatic reaction, it is also important to investigate how well a particular enzyme reacts. Enzyme kinetics is therefore measured by measuring the rate of enzyme action with reference to concentration of the substrate. In enzyme kinetics, we look at two parameters namely Vmax and Km. Vmax is the velocity at the highest possible level of substrate concentration while Km refers to Michaelis’ constant and it is the concentration of the substrate at which the rate of reaction is ½ Vmax. Through measurement of the rate of formation of the products at different concentrations of the substrate, we are able to measure the rate of enzyme activity. This study focuses on peroxidase and aims at measuring its kinetics and optimal pH Objectives To measure peroxidase enzyme kinetics To determine optimal pH for peroxidase enzyme Methodology Enzyme Preparation Peroxidase enzyme was prepared by blending 25g of radishes in 350 milliliters of ice-cold distilled water. The brei was then filtered through a cheese cloth before going through a centrifugation for five minutes at 500 x g and 4 minutes. The extract was then kept in ice. Determination of working enzyme concentrations Several different concentrations were assayed to determine the effect of enzyme concentration on the reaction rates and then determine the amount of extract to be used for the other parts of the experiment. Dilutions of 1x, 2x, 4x, 6x, 8x, 10x, and 20x were prepared in spectrophotometer tubes and kept in ice. 1 ml of water was then added to the first tube for each dilution. The blank was then used to adjust the reading of the spectrophotometer to read zero absorbance at 470nm for each of the six blanks corresponding to respective experimental reaction. The spectrophotometer tubes were then warmed to room temperature before adding 1 ml 40mM guaicol before mixing the content immediately by capping the tubes. The tubes were then placed in a spectrophotometer and absorbance read at 10 seconds for 1x and 2x tubes, at 20 seconds intervals for 4x and 6x tubes and 30 second intervals for 8x and 10x tubes. The data was then recorded in Table 1 as shown. The initial velocity for reaction for each tube were also calculated and expressed as change in A470/minute and the data recorded in Table 1 Effect of pH on Peroxidase Enzyme Eight spectrophotometer tubes containing the volume of water and extract that gave an enzyme activity rate of 0.1unit/minute in part B above (table1) were used in this experiment. 0.5ml of H2O2 and 0.5 of appropriate buffer were added to each of the tubes above before performing reaction at 8 different pH; 3 4, 5, 6, 7, 8, 9, and 10 and each receiving 0.5ml of a different buffer. The spectrophotometer was then zeroed using appropriate blank before adding and mixing 1ml 40mM guaiacol to the pH 3.0 tube. The contents in the tubes were then mixed and placed in the spectrophotometer and the absorbance read at suitable intervals and the readings recorded in table III. This step was the repeated for the remaining tubes one at a time. The initial velocity at each pH, was then expressed as AA470/minute and recorded in the table III. Enzyme Kinetics Six spectrophotometer tubes containing the mixture of enzymes, water, buffer and hydrogen peroxide that gave a reaction rate of 0.1unit/minute in part B were prepared. Spectrophotometer was then blanked and the contents of 50 microml guaiacol tube added to tubes of diluted enzyme. The contents were mixed and placed in a spectrophotometer and left until the needle started moving. The absorbance level was the read at suitable intervals and the data recorded in table IV. This procedure was repeated for each of the remaining tubes at a time before calculating the initial velocity of each substrate concentration. The results were then recorded in table IV. From the data from the table, Lineweaver-Burk plot of 1/v vs. 1/(S) and used to calculate Vmax and Km of guaiacol. Results Graph 1: The effect of Enzyme Concentration on enzyme activity The following graph shows the effect enzyme concentration on the rate of reaction at different dilutions Graph 2: Effect of pH on enzyme activity The following graph shows the effect of different pH levels on peroxidase enzyme Enzyme Kinetics Guaiacol Concentration (S) Initial Velocity (v) 1/(S) 1/(V) 50 0.005 0.02 200 150 0.016 0.0066 62.5 300 0.037 0.0033 27.0 1000 0.093 0.001 10.75 3000 0.117 0.00033 8.54 10000 0.2 0.0001 5 Graph 3: Lineweaver-Burk Plot The graph below shows namely Vmax and Km calculations from different substrate and absorbance rates. Discussions The results of this experiment shows that the performance of peroxidase enzyme is greatly affected by all the two factors that it was subjected to i.e. enzyme concentration and pH. While the effect of enzyme concentration can clearly be seen in graph1, it is important to note that the effect of pH on the enzyme activity does not show a predictive trend but a specific one. Peroxidase enzyme is highly active at pH 4 while averagely very low in other pH levels. This shows specificity of enzymes with respect to environmental factors. We can see clearly how increasing dilutions results into the reduction in enzyme activity for peroxidase. Low concentrations of enzymes affect activity since the rate of formation of substrate enzyme complexes is reduced. The results shows that by reducing concentration by half, we get velocity reduced by half from initial velocity of 1.17 to 0.83. This trend is evident throughout other dilutions thus we can conclude that enzyme concentrations is directly proportional to the rate of enzyme activity since reduction in concentration reduces enzyme activity and vice versa. Effect of pH on peroxidase enzyme in this experiment shows a very interesting scenario. The data derived from this experiment shows that peroxidase enzyme is highly sensitive to pH levels. Looking at initial velocity (V) at different pH levels, it is clear peroxidase enzyme is most active at pH 4 where we realized absorbance levels of 0.52. Other pH levels show very low levels of peroxidase enzyme activity. It is also worth noting that from pH 5-10, peroxidase activity remains almost the same. In the introduction of this report we outlined that enzymes are highly sensitive to the environmental factors thus can only work within specific favorable environments to produce desired products. As stated by Onzález (2014), the sensitivity of enzymes also accounts for their specificity. In the natural setting, enzymes are subjected to different environmental conditions within our internal environments. A good example is the presence of enzyme pepsin in our stomach at pH 2 while other enzymes will be denatured at this pH (Das et al 2011). It is therefore important to note that ph effect on enzyme is specific thus performance of enzymes too. We also noted that enzyme catalyzes reaction by physically attaching on the active sites of the substrate thus its shape is vital for performance. Moreover, we also noted in our introduction that pH affects enzyme structure thus making them very ineffective. Enzyme catalyses large amount of reactions without being consumed, thus if the concentration is dilute to the extent that large portions of substrate molecule do to get attached to “active site”, that particular chemical reaction will occur very slowly. On the other hand when the concentrations are high, most of the active sites are not used thus there will be high rate of reactions. However, since substrate is converted into products, the consequent reduction in substrate results into reduction in enzymatic reaction. Optimal concentration ensures that the rate of reaction is high and steady for a long period of time. It is therefore important to note that there is a vital importance of the concentration of both enzyme and substrate. Vmax and Km could not be determined due to the nature of the graph derived from the experiment. This was therefore calculated. Conclusions This experiment shows that peroxidase enzyme is specific to pH 4 and its performance is highly deteriorated at any other pH above or below. Concentration of enzyme however increases the rate of reaction with availability of substrate. However, optimal reaction can be attained with the availability of enzyme in excess of substrate. The optimal state is achieved when the rate of action is steady despite increase in substrate concentration. The range of the data collected was not able to derive Vmax and Km and could be due to the differences in absorbance or errors in reading. References Campbell, Neil A., Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson. (2008). Biology: Eigth edition. San Francisco: Pearson Benjamin Cummings. Das, B., Chakraborty, A., Ghosh, S., & Chakrabarti, K. (2011). Studies on the effect of pH and carbon sources on enzyme activities of some pectinolytic bacteria isolated from jute retting water. Turkish Journal Of Biology, 25(6), 671-678. Onteh, F. A., Grandison, A. S., & Lewis, M. J. (2005). Factors affecting lactoperoxidase activity. International Journal Of Dairy Technology, 58(4), 233-236. Onzález, L. J., Moreno, D. M., Bonomo, R. A., & Vila, A. J. (2014). Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-β-Lactamase Are Modulated by Second Sphere Residues. Plos Pathogens, 10(1), 1-12. Read More