Research on an Enzyme

Research on an Enzyme
I have carried out a research on the type of enzyme that I have chosen to use, urease. Urease is an enzyme that catalyses the hydrolysis of urea into ammonia and carbon dioxide. The reaction occurs as follows: (NH2)2 CO+3H2O CO2 +2NH4OH [image] It is a globular protein found in bacteria and several higher plants as found by James Sumner in 1926. Its molecular weight is 480kDa with optimum pH of 7.4 and its enzymatic specificity is urea and hydroxyurea. ================= Urea is the chief solid component of mammalian urine; synthesized from ammonia and carbon dioxide and used as fertilizer, in animal feed and in plastics. Preliminary Work I have carried some preliminary experiment to help me decide on how to measure measure the effect of temperature on the activity of urease. In the first experiment, I used the universal indicator to find out if temperature has on effect on the pH of the ammonium produced by the reaction. In the second experiment I used the litmus paper but it was still the same effect. I found out that it does have an effect but not a lot and it was hard to measure as the colour was always green although in different shades. In the third one I used the electronic pH indicator but the pH numbers hardly changed. I decided on using the electronic pH indicator but the pH numbers hardly changed.
I then concluded that the pH is hardly affectedby temperature so I decided to measure the amount of CO 2 produced, I carried out a preliminary experiment on this and it worked so this is what I am going to do in my experiment. In this experiment I found out that as the temperature increases above 40 Â? C the gas is expanded so I am going to allow 5-10 minutes of cool down before I record the results. plan aim To find out how temperature affects the enzymic action of urease on urea to produce Carbon dioxide and ammonia. Hypothesis My hypothesis is that as the temperature increases the rate of reaction also increases like many other non-enzymic chemical reactions. However enzymes are biological catalysts and they get denatured if the temperature rises above their optimum temperatures, which in this case is 65 Â? C. Enzyme denaturing is when the weak hydrogen bonds that maintain its quaternary structure have been broken, irreversibly changing the shape of the molecule and it can no longer function. This is due to the increase of kinetic energy increasing the collisions between the particles therefore breaking the bonds, as they are very delicate. I predict that the graph is going to look like this: This is because the temperature is going to continue to increase the enzyme reaction to the substrate until it reaches its optimum temperature of 65ËšC and then it will decrease sharply. As I have mentioned before this is caused by the enzyme denaturing. Urease, like other enzymes is a globular protein. Urease, like many other globular proteins has individual molecules with a complex quaternary structure. They are spherical or globular in shape and it is soluble in water. Proteins are made up of amino acids joined together by a condensation reaction to form a dipeptide bond as shown on the diagram below. A peptide is formed when the amino acids are joined into a small chain, a protein is the final, functional molecule made up of these peptide bonds. Therefore proteins are classified as complex molecules. They have a primary structure, the sequence of amino acids in the protein. Followed by a secondary structure, this is when they form their shape and patterns when they fold. This is to accommodate the hydrogen bonds in the most suitable arrangement. Hydrogen bonds hold in position the secondary structure of the protein and they are quite weak and unstable and therefore can be easily broken. The tertiary structure of a protein is its overall three-dimensional shape. The final one is the quaternary structure, which consists of more than one polypeptide. Apparatus Function
>6 water baths

>2 measuring cylinders

1 ? 100cm 2 ? 50cm ³
>7 thermometers

>2 beaker

>2 conical flasks

>1 stirring rod

>1 spatula

>1 measuring boat

>1 measuring scale

>1 stand

>1 gas collecting tube

>1 filter

>1 piece of filter cloth

>1 pipette

>1 stop watch

>For different temperatures

>To measure water when making concentrations.

>To measure urea and urease.

>To measure temperature.

>To put the urea and urease in.

>To mix urea and urease in.

>For stirring.

>To scoop urea and urease on the measuring boat when measuring them.

>To measure urea and urease on.

>To put urea and urease on when measuring.

>To hold the gas collecting tube in position.

>To collect the gas in.

>To filter urease

>To thoroughly filter urease

>For accuracy when making measurements of the urea and urease.

>To time the experiments

Apparatus, Chemicals and their Function Chemical Function
>Urea

>Urease

>Distilled water

>To react with urease to produce ammonia and carbon dioxide, this will enable me to how fast it is when reacted together

>To catalyse the hydrolysis of urea into ammonia and CO2.

>To make the urea and urease 5% concentrations

Hazards If swallowed: Wash out mouth and give a glass or two of water and seek medical attention. If solid gets into eyes: Flood the eye with gently running water and wash thoroughly with soap and cold water. If spilt in lab: Scoop up as much as possible. Wash area thoroughly with detergent and water. If spilt on skin or clothes: Remove contaminated clothing. Flood area with water and wash thoroughly with soap and cold water. Urea Inhalation: Causes irritation to the respiratory tract. Symptoms may include coughing, shortness of breath. May be absorbed into the bloodstream with symptoms similar to ingestion. Ingestion: Causes irritation to the gastrointestinal tract. Symptoms may include nausea, vomiting and diarrhoea. May also cause headache, confusion and electrolyte depletion. Skin Contact: Causes irritation to skin. Symptoms include redness, itching, and pain. Eye Contact: Causes irritation, redness, and pain. Chronic Exposure: A study of 67 workers in an environment with high airborne concentrations of urea found a high incidence of protein metabolism disturbances, moderate emphysema, and chronic weight loss. Aggravation of Pre-existing Conditions: Supersensitive individuals with skin or eye problems, kidney impairment or asthmatic condition should have physician?s approval before exposure to urea dust. First Aid Measures for Urea -???????????? Inhalation:Remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention. Ingestion:Induce vomiting immediately as directed by medical personnel. Never give anything by mouth to an unconscious person. Get medical attention. Skin Contact:Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Wash clothing before reuse. Thoroughly clean shoes before reuse. Get medical attention if symptoms occur. Eye Contact:Immediately flush eyes with plenty of water for at least 15 minutes, lifting lower and upper eyelids occasionally. Get medical attention immediately. Safety Precautions v Minimise contact or inhalation, as it is a potential allergen. It can cause asthma and/ irritate the membranes of the eyes and the nose. v Wear safety glasses as the urease dust may cause sight transient and irritation. v Do not eat or attempt to eat, as urease is harmful when ingested into the body in large quantities. v Do not inhale as inhalation of this enzyme may cause pulmonary sensitisation and may cause bronchiole spasms in asthma. v Wear a safety lab coat to avoid contact with your skin as this may cause sensitisation. v After the experiment wash your hands thoroughly with soap. v For hand protection from the enzyme wear rubber gloves to avoid contact. Distilled water is not harmful at all, it is just like normal water but the distillation process removes almost all impurities from water. Distillers are commonly used for removing nitrate, bacteria, sodium, hardness, dissolved solids, most organic compounds, heavy metals, and radio nuclides from water. Distillers remove about 99.5 percent of the impurities from the original water. Therefore I have chosen this type of water because it is more pure and allows for accuracy in the experiment. Method Preparation: 1. Collect apparatus 2. Set up the water bath at 30 � C, 40 � C, 50 � C, 60 � C, 70 � C and 80 � C. 3. Set up the stand, gas collecting tube and water bath as shown below: 4. Measure 5g of urease and 5g of urea separately using a spatula and the measuring boat. 5. Put them in separate beakers and label name of chemical and concentration. Measure 95cm³ of distilled water and put in the beakers. 6. Stir them thoroughly for 2-3 minutes each. Place the funnel on the measuring cylinder and place the filter cloth the funnel and filter the urease. Experimental Procedure 1. Pipette 20cm3 of urease into a conical flask and 20cm³ of urea into a 50 cm ³ -measuring cylinder and put next to the water bath-gas collecting tube set-up. 2. Set stopwatch at zero, place conical flask in water bath and add urea to urease and quickly close it using the cork of the gas collecting tube to capture the CO 2 gas and start the timer. 3. Stop the timer at 5 minutes and record gas collected. 4. Move apparatus to the next water bath and repeat the experiment. *When you get to 40 � C, allow a cool down of 3 minuets and add 2 minutes after an addition of 10 � C to the temperature. *When the experiment is over repeat 3 times. Results Temperature (�C) 20 30 40 50 60 70 80 Text Box: Experiments Volume of experiment 1 (cm ³) 2.50 7.90 25.00 33.00 43.50 7.00 1.50 Volume of experiment 2 (cm³) 2.10 11.00 23.60 30.65 45.80 3.80 3.20 Volume of experiment 3 (cm ³) 2.00 9.30 27.20 32.50 40.50 5.65 1.90 Average (cm ³) 3.30 9.40 25.27 32.05 43.27 5.48 2.20 Analysis Temperature Average volume (cm³) 20 3.30 30 9.40 40 25.27 50 32.05 60 43.27 70 5.48 80 2.20 [image] As from the graph and figures above, it shows that my prediction was right. As the temperature increases the enzyme produces more carbon dioxide gas until it gets to an optimum temperature of which in this case it is 60�C and as shown on the graph the carbon dioxide release rate decreased rapidly as the enzyme urease becomes denatured above this temperature and it will not work as effectively. This must mean that the rate of reaction increases, as the temperature increases until it gets to the optimum temperature and then it will start to decrease. This information can be shown by the graph I am going to produce below. Calculating the rate of reaction: 60secs � 5 = 300secs 3.30cm³ � 300secs = 0.01 9.40cm³ �300secs = 0.03 25.27cm³ �300secs = 0.08 32.05cm³ �300secs = 0.11 43.27cm³ �300secs = 0.14 5.48cm³ �300secs = 0.02 2.2 cm³ �300secs = 0.01 Temperature (cm³) Rate of Reaction 20 0.01 30 0.03 40 0.08 50 0.11 60 0.14 70 0.02 80 0.01

Research on an Enzyme 7.1 of 10 on the basis of 1982 Review.