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Investigate the effect of pH on Trypsin

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Introduction

Biology Coursework Plan Aim Investigate the effect of pH on Trypsin Prediction / hypothesis * pH will affect trypsin action * as pH increases, tryrpsin will show increasing activity up to an optimum pH * the action of the enzyme trypsin on the substrate egg albumen will be at a maximum at an optimum pH of around 7 (neutral) to 8 (slightly alkaline) see later about pH of duodenum * as pH continues above this, trypsin activity will decrease Background / Introduction Proteins are complex organic compounds consisting of amino acids joined by peptide bonds which form highly folded three dimensional or tertiary structures. The bonds that maintain the tertiary structure of the protein are a result of interactions between the R groups of the amino acids: disulphide bridges (strong covalent), hydrogen (weak) bonds, ionic or electrovalent bonds, hydrophobic interactions. Enzymes, such as trypsin, are globular proteins with a specific shaped active site into which the correct substrate can fit. trypsin protein / polypeptide peptides Trypsin is a protease enzyme: a hydrolytic or digestive protein that cleaves peptide bonds. It is produced in the pancreas in the form of trypsinogen, and is then transported to the duodenum of the small intestine, where the digestion of proteins to polypeptides and amino acids begins. The pH of the duodenum is alkaline, due to the presence of Bile containing alkali sodium hydrogen carbonate to neutralise the stomach acid. ...read more.

Middle

The colorimeter should be tested and used to confirm that absorbancy readings decrease with the progressing reaction. The correct coloured filter should be determined it needs to absorb in the parts of the spectrum opposite to those that the solution absorbs, so it only lets through that wavelength light. Blue is probably the best as the egg albumen with the enzyme will be a yellowish tint. Apparatus ( x 3 for three replicates): 14 test tubes 14 cuvettes trypsin solution boiled trypsin solution distilled water albumen / egg white solution buffer solutions pH 1 - 12 water bath - 40oC 1 cm 3 and 2 cm 3 pipette and pipette fillers colorimeter, filter, reference cuvette marker pen glass rod Risk Assessment A lab coat and goggles should be worn at all times during the experiment as they will minimise any substance contact with the skin or eyes in the event of a spillage. All enzymes, are potential allergens and have especially potent effects if inhaled as a dust, thus the trypsin will be kept in aqueous form and will be handled wearing gloves at all times. Glassware should be handled carefully at all times and test tubes should not be raised above shoulder level. Special care should be taken with the electronic colourimeter, which to prevent electric shock should not be operated near any water source (water bath, sink) ...read more.

Conclusion

5. Pour the solution into the cuvette up to the pre-indicated fill mark (4 cm 3 ) and place it into the colorimeter sample chamber with the clear sides in line with the light path, making sure not to touch the clear sides and in doing so leave fingerprints that may interfere with the reading. 6. Obtain an absorbancy reading for time 0, remove the cuvette from the colorimeter and place back into a holder in the water bath. 7. Record the changes in colour detected by the instrument at 30 second intervals for 10 minutes. 8. Repeat for each pH and the two controls. 9. Tabulate results and then plot a graph of absorbance units against time (minutes) for each pH. At the optimum pH, to start with, the absorbance reading should decrease rapidly as the rate of enzyme action should be high and then the decrease will begin to slow as most of the substrate (albumen) is used up in the reaction. During the linear portion of the curve, calculate the initial rate of reaction for each pH by finding the gradient: take a tangent and divide the y (absorbance) value by the x (time) value. 10. By plotting the values for the rates of reactions against the corresponding pH the graph you produce should resemble a bell shaped curve, with the maximum / peak rate at the enzyme's optimum pH. For most enzymes this is about pH 7-8 as it is the normal pH within the cell. ...read more.

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A well written plan for an investigation. The author has a clear understanding of the relevant theory but in places more detailed explanations would help to demonstrate this. Variables could be more fully discussed.

Marked by teacher Adam Roberts 05/09/2013

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