PRACTICAL 5 AND 6: PURIFICATION OF BIOMOLECULES

BIOL I 1100


In this practical Gel Permeation Chromatography, also known as size exclusion chromatography was used to separate a mixture of small and large biomolecules according to their size. In GPC the matrix contains tiny gel beads which have an open structure. The large molecules will travel faster through the gel, this is due to the fact that the small molecules can enter the gel beads which contain a gel matrix that encloses an internal solvent space whereas the large molecules cannot.
In this case the mixture contained trypsin (protein), glucose (carbohydrate), and cholesterol (lipid). Twenty 2 mL fractions were collected as the solution run through the gel together with a running buffer which keeps the beads hydrated. The larger molecules will come out first, whereas the smaller molecules will come out later because they have to travel through a bigger volume of solvent. Blue Dextran and yellow pNP were used as size markers, the blue colour came first indicating the molecules of high molecular weight and the yellow colour came last indicating those molecules with low molecular weight.
Trypsin and glucose are soluble in the running buffer solution but cholesterol is not, cholesterol should be extracted by a liquid-liquid extraction, which it wasn’t carried out. On the other hand, Protein can be detected using Bradford reagent which is a dye binding method, when protein is mixed with the Bradford reagent there is a change in colour of the reagent. The intensity of the colour, in this case blue, being proportional to the amount of protein. The enzyme glucose oxidase was used to detect the presence of sugar, again the intensity of the colour, in this case red, would indicate the presence of sugars. The following table of results was obtained when adding Bradford reagent and Glucose oxidase to twenty 100µl fractions prepared using the previously separated 2mL fractions.

The minus symbol represents no colour, whereas the + symbol represents intensity being +++ the maximum intensity. In the case of protein the colour developed was blue whereas in glucose it was red.
The results show how Trypsin which is a large molecule with a complex 3D structure, comes out at an earlier stage because it is unable to enter the beads and therefore passes through the column at the same speed as the buffer. On the other hand glucose, a monomer with low molecular weight, came out at a later stage because it is a smaller molecule which is able to enter the beads, and therefore has to travel trough a larger volume of solvent.
If a plot of the concentration of the concentration of trypsin and glucose was to be made it would look like the graph below, indicating the separation of the two components.