Features of Forensic Science: Tyrosinase Practical - Research Proposal Example

FORENSIC SCIENCE: TYROSINASE PRACTICAL and Table of Contents Introduction 3 Hypothesis 5 Methodology 5 Materials for data collection 5 Procedure 5 Results 6 Discussion 13 Conclusion 13 Bibliography 15 Introduction Tyrosinase is a copper-containing enzyme which is widely distributed in plants, microorganisms, and animals (Axford, 2003). It is found inside the melanosomes. In humans, tyrosinase is encoded by the TYR gene. TYR gene is located in the melanocytes and gives instructions for making the tyrosinase enzyme. This catalyzes the production of melanin and other pigments by oxidation from tyrosine of melanin and other pigments. In molecular biology, it refers to an oxidase that is a rate-limiting enzyme used in controlling the production of melanin. Melanin is a substance that gives the hair, eyes, and skins their colour. This is also found in the light-sensitive tissue at the retina part of the eye where it plays a role in normal vision. Tyrosinase is the first step in the production of melanin. This converts a protein building block or amino acid called tyrosine into dopaquinone (WASSARMAN, 1998). Tyrosinase is mainly involved in two melanin synthesis reactions, that is, firstly it is involved in the hydroxylation of a menophelol. It is also involved in the conversion of an o-diphenol to the corresponding o-quinone. o-Quinone goes through a number of reactions in order to form melanin. Tyrosinase inhibitor has an ambiguous meaning in research articles. Some authors use the terminology in terms of melanogenesis inhibitors that mainly focuses on decreasing the formation of melanin regardless of any direct interaction between enzyme and inhibitor. They have become a great concern because of the role of tyrosinase in both mammalian melanogenesis and fungi or fruit enzymatic browning. Melanogenesis is the process that leads to the formation of melanin. Melanin plays a crucial role in protecting the human skin from harmful effects of UV radiation from the sun (Freinkel, 2001). Tyrosinase inhibitors are widely used in cosmetics and dermatological treatments. They can be derived from both synthetic and natural sources. Many potential tyrosinase inhibitors are examined in the presence of L- or L- tyrosine. When these phenolics exhibit an excellent affinity for tyrosinase, dopachrome formation is prevented. For this reason, they could be regarded as being tyrosinase inhibitors (AMERICAN PEPTIDE SYMPOSIUM, & BLONDELLE, S, 2006). Nonspecific tyrosinase inactivators such as, bases and acids which denature the enzyme and inhibits its activities are usually mistakenly referred to as tyrosinase inhibitors. The tyrosinase inhibitors could be classified into four types, including the competitive inhibitors, uncompetitive inhibitors, non-competitive inhibitors, and mixed type. A competitive inhibitor combines with a free tyrosinase which prevents substrate binding. A competitive inhibitor can be a tyrosinase substrate analogs, copper ion chelator, L-DOPA, or derivatives of L-tyrosine (STARR, 2009). An uncompetitive inhibitor only binds to the tyrosinasesubstrate complex. It can bind to a free tyrosinase and a tyrosinase-substrate complex, with the same equilibrium constant. The tyrosinase inhibitor strength is normally expressed as the inhibitory IC50 value (AMERICAN PEPTIDE SYMPOSIUM, & BLONDELLE, S, 2006). This is the concentration that an inhibitor needs to inhibit half of the enzyme activity, in a tested condition. A mixed type consists of the competitive and uncompetitive inhibitors mixed and bound with the tyrosinase-substrate complex and with a free tyrosinase. For most of the mixed-type inhibitors, the equilibrium binding constants for the tyrosinase-substrate complex and the free tyrosinase are different. Forensic Science or Forensics is the application of science to law in order to help in the interpretation of clues in crime investigation (TILSTONE, 2006). Forensic science is mostly used in investigating criminal cases that involves victims such as rape, robbery, kidnapping, and murder. One way of forensics especially in cases where there are unidentified people is using human appearance information that can be obtained from DNA of crime scene samples. Human appearance prediction is very useful in solving forensic and cold cases where there are no suspects identified by other conventional investigation methods, where the victims are too mutilated to be recognized or where the suspects are too many. Tyrisinase is used in forensic science by helping in the development of fingerprinting that is useful in the process. Hypothesis From the readings, this research has come up with the following hypothesis; Hypothesis 1. When the enzyme is kept constant and the substrate concentration is increased gradually, the velocity of the reaction will increase until it reaches maximum. Hypothesis 2. Benzoic acid and thiourea are competitive inhibitors of tyrosinase. Methodology Materials for data collection In order to collect data an experiment was conducted where a general assay procedure was done. Materials used in this experiment include phosphate buffer, cuvettes, inhibitor, enzymes, pipette, colorimeter, parafilm, stopclock, and substrate. To be able to determine the inhibitory effects of Benzoic acid and Thiourea, materials used include; enzyme, buffer, inhibitor, L-DOPA, and tyrosinase. Procedure The general assay procedure included gathering all the materials required in the experiment. The required volumes of phosphate buffer, substrate, and inhibitor were pipette in a cuvette. These were mixed well and then the colorimeter was zeroed on the cuvette. The cuvette was then removed from the colorimeter. The required volume of enzyme was quickly added. This was mixed and the stopclock was started. The cuvette was placed on the colometer and the first reading was taken after 15 seconds. This reading was continued at intervals of 15 seconds for 2 minutes. Results were recorded in tables and analysis was done based on these results. Results From the experiment done, below is the result found Part A Absorbance Time (Seconds) 15 30 45 60 75 90 105 120 Tube 1 0.02 0.025 0.03 0.04 0.05 0.06 0.07 0.08 Tube 2 0.04 0.06 0.08 0.09 0.11 0.13 0.14 0.155 Tube 3 0.04 0.08 0.11 0.15 0.18 0.21 0.23 0.26 Tube 4 0.09 0.16 0.23 0.29 0.36 0.41 0.46 0.51 Graph A Gradient= Change in Y ÷ Change in X Tube 1: (0.08- 0.02) ÷ (120-15) = 0.0006 Tube 2: (0.155- 0.04) ÷ (120-15) = 0.0011 Tube 3: (0.26- 0.04) ÷ (120-15) = 0.0021 Tube 2: (0.51- 0.09) ÷ (120-15) = 0.0040 Velocity= 1 ÷ absorbance per minute Tube 1: 1 ÷ 0.0006= 1666.7 Tube 2: 1 ÷ 0.0011= 909.1 Tube 3: 1 ÷ 0.0021= 476. 2 Tube 4: 1 ÷ 0.0040= 250 1/ [S] values 1mgml-1 Graph of 1/ [S] against 1/V The results above shows that when the enzyme is kept constant and the substrate concentration is increased gradually, the velocity of the reaction will increase until in reaches maximum. After that point the increase in the substrat concentration will not increase the velocity. Part B Absorbance Time (Seconds) 15 30 45 60 75 90 105 120 Tube 1 0.09 0.16 0.24 0.30 0.36 0.42 0.47 0.52 Tube 2 0.07 0.13 0.18 0.25 0.30 0.35 0.40 0.45 Tube 3 0.05 0.10 0.14 0.18 0.23 0.27 0.31 0.35 Tube 4 0.05 0.09 0.13 0.16 0.20 0.24 0.27 0.31 Tube 5 0.04 0.07 0.11 0.14 0.17 0.20 0.23 0.26 Tube 6 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Graph B Gradient= Change in Y ÷ Change in X Tube 1: (0.52- 0.09) ÷ (120-15) = 0.004 Tube 2: (0.45- 0.07) ÷ (120-15) = 0.004 Tube 3: (0.35- 0.5) ÷ (120-75) = 0.004 Tube 4: (0.31- 0.05) ÷ (120-15) = 0.002 Tube 5: (0.26- 0.04) ÷ (120-15) = 0.002 Tube 6: (0.01- 0.01) ÷ (120-15) = 0 Velocity= 1 ÷ absorbance per minute Tube 1: 1 ÷ 0.004= 250 Tube 2: 1 ÷ 0.004= 250 Tube 3: 1 ÷ 0.004= 250 Tube 4: 1 ÷ 0.002= 500 Tube 5: 1 ÷ 0.002= 500 Tube 6: 1 ÷ 0.002= 0 1/ [S] values 1 ÷ 105 = 0.01 Part C Absorbance Time (Seconds) 15 30 45 60 75 90 105 120 Tube 1 0.04 0.08 0.11 0.15 0.18 0.21 0.25 0.28 Tube 2 0.04 0.07 0.11 0.14 0.17 0.20 0.23 0.26 Tube 3 0.04 0.08 0.12 0.15 0.18 0.21 0.24 0.27 Tube 4 0.04 0.07 0.11 0.14 0.16 0.18 0.21 0.23 Tube 5 0.04 0.07 0.09 0.12 0.14 0.16 0.18 0.20 Tube 6 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Graph C Gradient= Change in Y ÷ Change in X Tube 1: (0.28- 0.04) ÷ (120-15) = 0.002 Tube 2: (0.26- 0.04) ÷ (120-15) = 0.002 Tube 3: (0.27- 0.4) ÷ (120-75) = 0.002 Tube 4: (0.23- 0.04) ÷ (120-15) = 0.002 Tube 5: (0.20- 0.04) ÷ (120-15) = 0.002 Tube 6: (0.01- 0.01) ÷ (120-15) = 0 Velocity= 1 ÷ absorbance per minute Tube 1: 1 ÷ 0.002= 500 Tube 2: 1 ÷ 0.002= 500 Tube 3: 1 ÷ 0.002= 500 Tube 4: 1 ÷ 0.002= 500 Tube 5: 1 ÷ 0.002= 500 Tube 6: 1 ÷ 0.002= 0 Discussion Enzymes act as catalysts to proteins that speed up the rate of chemical reactions without being used up in the process. From the experiment, it is clear that the work of enzymes can be influenced by a number of factors that include the presence of inhibitors. The Enzymes can be inhibited competitively and noncompetitively. In the presence of a competitive inhibitor, a higher substrate concentration is required to achieve the same velocity that would have been reached in its absence. The results from tube C shows the presence of competitive inhibitors. With non-competitive inhibitor, enzyme molecules bound by the inhibitor are taken out of the game. Other factors that influence the work of enzymes include the concentration of substrate molecules [S] where the higher the concentration, the quicker the enzyme molecules collude and bind with them. From the experiment, the velocity increases the reduces as time increases, this shows another factor that affects enzymes. As the temperature, the motion of the molecules increases, however, enzymes are proteins which mean that they reach an upper limit where the enzyme becomes denatured and ineffective. Bibliography AMERICAN PEPTIDE SYMPOSIUM, & BLONDELLE, S, 2006. Understanding biology using peptides proceedings of the Nineteenth American Peptide Symposium. New York, N.Y: Springer. Axford, J., 2003. Glycobiology and Medicine. Volume 3 ed. New York, NY : Kluwer Acad. Freinkel, K. &. Woodley. T., 2001. The Biology of the Skin. s.l.:CRC Press. STARR, C., 2009. Biology: the unity and diversity of life.. Belmont, CA., Brooks/Cole: Cengage Learning. . TILSTONE, W. Et. Al., 2006. Forensic science: an encyclopedia of history, methods, and techniques. Santa Barbara, Calif: ABC-CLIO. WASSARMAN, P., 1998. Advances in developmental biochemistry. Volume 3 ed. Greenwich, Conn: JAI Press. Read More