The aim of this investigation is to investigate the affect of bile salts on the action of the enzyme lipase

Bile salts are secreted from the liver in the gall bladder, and then passed through the bile duct into the small intestine when food is passing through; they are involved in the emulsification of fats in the intestine, helping in the digestion and absorption of lipids. They also carry waste products to be excreted such as bile pigments. These derive from the breakdown of red blood cells. Bile salts are made up of a bile acid and an associated cation, which are usually amino acids. Bile salts are made up of two parts these are hydrophobic and hydrophilic components.Figure one:The above figure portrays the structure of bile acids.

The hydrophobic part of the molecule associates with particles of dietary fat which causes the fat globules to break down into microscopic droplets; this increases the surface area of the fats for digestion by lipases. While the hydrophilic part associates with water which in turn emulsifies the insoluble fat, which is then absorbed through the intestinal wall. The figure illustrated below shows the action of bile salts in emulsifying fats in the intestine.Figure one:The bile acids are made from cholesterol, which is either ingested as part of our diet or derived from hepatic synthesis, which are then conjugated to an amino acid. After the absorption and the emulsification process bile salts are separated from the dietary lipid and are recycled as they return back to the liver for reuse. Bile consists of 98% water, 0.8% bile salt, 0.7% inorganic salts, 0.

6%cholesterol and 0.2% bile pigments. In this investigation the presence of bile salts play a crucial role in the experiment as they emulsify the lipids making it easier for the lipase to act on the fats. The bile salt required is of 0.5 moles.Lipase as an enzyme:A catalyst is a substance that can speed up the rate of a chemical reaction without being altered or used up.

Catalysts allow substances to react more easily and quickly by providing an alternative pathway for the reaction and by helping bonds to break more easily, so the particles will need less energy to react and therefore speeding up the reaction. One known type of catalyst is enzymes. Enzymes are globular proteins, which act as biological catalysts. They are found in many kinds of cells, and collide into the precise 3-dimensional shape, consisting of long polypeptide chains folded into a tertiary structure. This structure is made stable by hydrogen, ionic and covalent bonds, in addition to disulphide bridges. In this investigation I will be using the enzyme lipase; the most important lipase is pancreatic lipase, which hydrolyses primary ester bonds into monoglycerides and free fatty acids.

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This lipase is strongly inhibited by bile salts, but is reactivated by colipase (colipase is secreted as procolipase, which is activated by trypsin during formation of colipase when needed).For a chemical reaction to take place, energy is required to bring the reactants’ molecules together in order for them to collide. However, for a more successful collision between the molecules and a faster rate of reaction, enzymes are introduced, as they can increase the chances of these molecular collisions. They do this by operating on a lock and key mechanism, shown below.

Figure 1: A diagram of the lock and key modelThis mechanism shows the substrate, which in this case is full fat milk, combining with the enzyme molecule, lipase, by attaching itself accurately onto a small part of the enzyme, known as the active site. The substrate (milk) is held in the active site by temporary bonds forming a structure, which is referred to as the enzyme-substrate complex.The energy needed to allow such a reaction to continue, is called the activation energy. Indeed, to increase the rate of reaction and the formation of new products, this energy must also be increased to bring the reactants closer together for collision, as shown in figure 2.1.

Figure 2.1Adding Enzymes on the other hand, decreases this activation energy, and at the same time increasing the collision between the reactants, hence increasing the rate of reaction, as seen in figure 2.2Figure 2.

2Lipase on lipidsA lipid is defined as an organic molecule that has a much greater solubility in common solvents (e.g. chloroform, ether) than in water. The enzyme lipase assists in the breakdown of lipids into smaller molecules known as carboxylic acid or fatty acids.

With the production of fatty acids the pH of the solution should change to a more acidic value, this can be detected by a decrease in the pH value. The production of the fat will come in contact and be emulsified by the presence of bile salt.TriglyceridesThe first step of fat absorption is hydrolysation of long-chain triglycerids, and thereafter emulsification by bile salts into micells and liquid cristals. These particles diffuse into the enterocytes, where they are resynthesised into triglycerides. The triglycerides are packed into chylomicrons and transported via the lymph.

In this investigation we will focus on the digestion of these triglycerides as they are first emulsified, and then broken down by lipase before transported in our body through the lymph. Triglycerides are the most common type of lipids; they are made up of three fatty acids and combined with one glycerol molecule in a condensation reaction. Fatty acids are blocks of two types of lipids – storage fats and the structural phospholipids. Fatty acids possess a long hydrocarbon chain with a carbonyl group attached to its tail. These chains can be described as saturated or unsaturated.

The latter is represented by double bonds between its carbon atoms, which suggests they lack the maximum possible amount of hydrogen, consequently, allowing them to melt more easily. Fatty acids differ from each other by the length of the hydrocarbon chain, and the number and position of double bonds. Below you can see a diagram of a saturated fatty acid, containing the maximum amount of hydrogen, and an unsaturated fatty acid with a double bond in the tail, creating a kink as the tail becomes bent, lacking the full number of hydrogen molecules.

Figure 3: the structure of a saturated and unsaturated fatty acid.Sodium carbonateSodium carbonate is used to reduce the acidity of chemicals, it is therefore maintains a very high pH which provides its alkali features. The sodium carbonate may be added to milk, before bile salt is in contact with the lipids, increasing the milks pH from neutral to alkali, this is necessary for when the enzyme is added one can identify the extent to which the pH drops again.HypothesisI predict that the greater the factor of bile salt the easier and quicker it is for the lipase to breakdown the lipids, whether or not this is correct will be observed by the duration taken for the pH to drop becoming more acidic once the bile salt has reacted with the cream emulsifying the fats thereby allowing the lipase to work more efficiently breaking down the lipids into single fatty acids.

Furthermore, once you decrease the concentration of bile salt the less powerful the activity of the lipase. This is a result of a diluted solution of bile restricting the degree of emulsification on the lipids therefore, lipids surface area does not increase dramatically leading to a slower reaction as the lipase requires a longer period of time to work on the fats, this means the decrease in pH will be limited and a longer period will be required for the pH to drop. One explanation for the reasons of this is that a limited surface area will mean less contact between substrate and enzyme, in this case the amount of collisions between the lipid and the lipase particles becomes less frequent.Null hypothesisThere is no difference between the varying concentrations on the activity of lipase. Furthermore the rate of reaction will not be affected by the presence of bile.VariablesThere are a number of variables, which must be controlled at all times throughout the experiment to ensure that my results are reliable and accurate, in addition to clarifying a fair test was conducted. The controlled variables are presented in the table below:Controlled variablesReasons why it must be controlledHow it will be controlledVolume of bile saltIt is important that the volume of bile salt added in the experiment remains the same in all trials and for all concentrations regardless of the change in bile concentration.

The reason for this, is in order to obtain accurate results reflecting that the concentration affected the reaction and not the volume of bile.10ml of bile salt will be measured out using a �0.05 pipette, for each trial and for each concentration.

Volume of lipaseIncreasing the volume of lipase will result in an increase in the rate of reaction as more frequent collisions will occur between the substrate (lipid) and enzyme (lipase), as the substrate molecules will bind to active site of lipase more often. Consequently, the volume of lipase must be maintained, as it is the concentration of bile salt I am testing. To ensure my results show the affect of the concentration of bile I must assert that the amount of lipase is kept constant throughout, clarifying that lipase has not affected my results, thereby avoiding inaccurate and unreliable results.10ml of lipase will be measured out using a �0.

05 pipette, for each trial and for each concentration.Volume of milkIncreasing the volume of milk, will result in more frequent collisions, due to a greater number of substrate binding more often with the active site of lipase. If this occurs, my results are unreliable as the data collected will be influenced, and affected by the amount of milk or lipids used in each experiment, therefore, disregarding the varying concentration of bile salt.

10ml of milk will be measured out using a �0.05 pipette, for each trial and for each concentration.Bile saltThe volume of bile salt must be controlled throughout although the concentration differs.

This is vital so that I may gain fair and accurate results. If I increase the volume of bile salt in one of the experiments, I will not be detecting the concentration of bile salt on the activity of lipase but the volume of bile salt on the activity of lipase.10ml of bile salt will be measured out using a �0.

05 pipette, for each trial and for each concentration.Duration once recording resultsI must ensure that this variable is controlled to avoid an interference with the rate of reaction. The longer the time period the lower the pH becomes. It is therefore necessary to maintain a constant duration throughout all the experiments enabling me to obtain accurate and fair results.All results will be recorded for every second for five minutes.This experiment does not only consist of controlled variables but also uncontrolled variables will be present, as illustrated below:Non-controlled variablesReasons why it will not be controlledHow I will ensure it is non-controlled variableConcentration of bile saltThe aim of this experiment is to examine the affect of different concentration of bile salt on the activity of lipase.

In order for this investigation to be carried out, I must change the concentration of bile salt observing the affect it has on the reaction.Refer to table one concentration of bile salt.Sodium hydroxideIf I had decided to place a fixed amount of sodium hydroxide then the pH of my solution (once everything is mixed together) may not always be the same.

Therefore, to ensure fair testing it is crucial that I keep adding drops of sodium hydroxide until the data logger reaches to pH10.Keep on adding drops of sodium hydroxide using a �0.05 pipette, for each trial and for each concentration, until data logger reaches to pH10.PreliminaryThis stage of my coursework was conducted to ensure that I chose the best and most accurate method for my actual experiment.

The first preliminary experiment held, was to investigate the amount of alkali solution used. At first we used 20cm of sodium hydrogen carbonate however, we observed that once this was added to the milk, it would instantly denature the milk before the lipase and bile salts were even added. This lead me to believe that I needed to decrease the amount of sodium hydrogen carbonate used; as a result I began using 15cm of the chemical, nevertheless the same outcome was witnessed and the milk denatured. The cause of this may have been due to the sodium hydrogen carbonate itself which I found to be extremely alkali. An alternative explanation would be due to a large amount of sodium hydrogen carbonate used, therefore denaturing the milk prohibiting a reaction as the bile salt and lipase were unable to act on the lipids, which became evident as the pH did not drop or change from pH10.

Consequently, I decided to use sodium carbonate which is not as alkali as the sodium hydrogen carbonate. Moreover, I decided that I did not require a large fixed volume of the chemical but in fact only droplets needed to be added until the pH reached to pH10, the amount of droplets varied in different experiments, this may have been due to the age of the sodium carbonate of perhaps a result of the particles settling in the solution.My second preliminary investigation was to acknowledge the affect of temperature on the rate of reaction allowing me to decide on the best temperature to use for the experiment, which will enable me with reliable and clear results. Enzymes work best at around 37�C; this is because the molecules become excited as they receive more heat, thereby more kinetic energy forcing them to collide more frequently with the substrate. However, above the optimum temperature the enzyme will denature due to the breaking of bonds and therefore no reaction can be examined. As a result, I decided to monitor the effect of the temperature on the reaction of the experiment; I therefore compared the rate of reaction under 18�C and 35�C. Given below are the results obtained for these two temperatures:18�CTime (sec)pH07.0907.

01506.8From the data above it is clear that only a slight change in pH was experienced when the lipase and bile salt were heated to 18�C.35�CTime (sec)pH07.0906.51506.4The table above indicates that once you increase the temperature, a greater decrease in pH will be shown. In this case once we increased the temperature from 18�C to 35�C the bile salt emulsified the lipids at a faster rate allowing the lipase to break them down at a quicker rate due an increase in collisions between the triglycerides and enzyme.

Subsequently, I will heat my lipase and bile salt together at 35�C in order to gain a greater decrease in pH implying a faster reaction on the break down of lipids.The final preliminary experiment held, was to explore which chemical of substrate would be best to use. We firstly tested out full-fat milk (substrate) and added sodium carbonate and lipase and bile salt. From this we received acceptable results as there were sufficient lipids present for the lipase to act on. However, when we used full-fat cream, we obtained better results, as there were more fats present therefore more substrate which meant more frequent collisions between enzyme and substrate particles.Preliminary methodFor my preliminary method I will demonstrate how I conducted and prepared my practice investigation for the different temperatures of 18�C and 35�C:1.

Prepare equipment neatly on a table. Attach labels on all apparatuses stating what each one contains to avoid confusion. Make sure all equipment is set out clearly and neatly.2. Before measuring out the chemicals you must shake the solutions well as occasionally the particles settle leading to inaccurate results. This can simply be done by shaking the beakers or flasks gently which contains the solutions ensuring nothing spills.3. Measure out 10ml of full fat cream using a pipette and add into a beaker labelled cream.

You must ensure that you wash the pipette as it will be required once measuring other substances.4. Add sodium carbonate into beaker labelled sodium carbonate using a pipette; make sure the pipette has been washed out so that the sodium carbonate does not react with anything before the experiment begins, this will result in accurate and precise results.5.

Measure out 10ml of lipase into test tube labelled lipase using pipette again make sure it has been rinsed out of any chemicals.6. measure out 10ml of bile salt containing a concentration of 3% (3% should be used as this is the median concentration of bile using pipette, and add test tube with label stating what it is.7.

Boil some water in kettle then pour water into a large beaker (1000ml)8. Add test tube labelled lipase and bile salt into beaker and monitor with thermometer until they reach 18�C.9. Now add lipase and bile with the cream in the beaker labelled cream.10. Place probe into the beaker of your mixture.11.

Using a burette add drops of sodium carbonate into beaker, until pH reads 10.0pH.12. You are now ready to record your results:13. Immediately start recording your graph.

14. Once the above has been maintained, and the graph is complete repeat the above steps but keep your test tubes containing lipase and 3% bile in the beaker until they reach 35�C.Apparatuses requiredThere are a number of equipments required in order to perform this is experiment, each apparatus possesses a specific job and is used as it enables the outcome of the most accurate and reliable results.Apparatus usedUse of apparatusReason for choiceTest tubeUsed to place different concentrations of bile salts, also used to place lipase to heat.The test tubes contain small surface this ensures that little quantity of bile or lipase is lost.

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