Investigating the effect of a chosen factor on the activity of Lipase

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Investigating the effect of a chosen factor on the activity of Lipase

Aim:

To explore the action of Lipase and Bile salts on lipids at different temperatures, and assess their roles in digestion.

Theory, Hypothesis and Prediction:

In nutrition, intake of lipids is necessary to form a source of energy storage, i.e. Fats. But as the lipid molecule is highly hydrophobic, it needs to be broken down first to fatty acids and glycerol to be taken in efficiently.

As the diagram? shows, there are three fatty acid tails

present in the lipid molecule, so it can diffuse quite

freely through the plasma membrane, yet it needs to be

broken down nonetheless so that it can be taken up

readily through the walls of the digestive tract, to be

transported to the areas of the body where it is used to form lipids again, for either:

* Deposition as fat (energy storage)

* Formation of cholesterol

* Formation of steroid hormones (e.g. testosterone)

* Formation of phospholipids for plasma membrane construction, or

* Immediate respiration as energy molecules.

The break down of lipids into their constituent molecules is a four-stage process and begins in the stomach and continues into the small intestine: -

. Secretion of Bile salts and Lipases into the stomach,

2. Emulsification of Lipids with the Bile salts

3. The enzymatic hydrolysis of the ester linkages in the triglycerides with the Lipases

4. The formation of lipid containing bile salt micelles which transport the lipids to the cells for resynthesis or oxidation

The use of Bile salts is required because lipids form non-polar structures that are insoluble in water. As water is the main medium of digestion in the lumen, this will pose some difficulty as the water soluble enzymes will not be able to digest the lipids efficiently unless they are somehow broken up in the water mechanically so that there is greater surface area to react with. The Bile salts

accomplish this by emulsifying the lipid so that the

enzymes will be able to react with them much more

easily and rapidly (diagram opposite?) As

derivatives of cholesterol, bile salts have both

hydrophilic and hydrophobic domains (amphipathic). On exposure to triglycerides, the hydrophobic portions of bile salts intercalate into the lipid, with the hydrophilic domains remaining at the surface. Such coating with bile salts aids in breakdown of large droplets into smaller and smaller droplets. This of course is important for the lipases, as now the lipids are in a form that can be relatively easily broken down chemically. The lipases do so by binding with the ester linkage at a special site known as the active site, which has a specific shape that will only fit that portion of the molecule - it is specialised. This is known as the lock and key hypothesis. At this site, the ester linkage is hydrolysed by addition of water, and the outer two fatty acids break off. By further action of bile, still tinier droplets called micelles are formed which are polar and consist of bile and bile salts, monoglycerides, fatty acids and glycerol. Once the absorptive cells of the intestines have absorbed these products, they are again converted to triglycerides that together with phospholipids from protein-coated droplets known as chylomicrons, which are the medium they are transported as.
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To detect the progression of the reaction in this experiment is a little difficult. However the fact that fatty acids are produced during the breakdown of Lipids can be used to provide a crude measure of the progression of the reaction. This is why Phenolphthalein indicator will be used. Above pHs of around 10 the indicator is pink, but below pH 8.4 it is colourless. So, if this indicator is added to a tube containing an alkaline sample of lipid being hydrolysed, it should become colourless, as the fatty acids that accumulate should neutralise any alkali in solution. ...

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