Sunday, 26 July 2009

The experiment of Protein Assay introduces us methods of determining protein concentration. We are using the Lowry Assay using absorbance at 750 nm.

We are using the spectrophotometer to determine the absorbance of the protein. The Lowry method depends on the presence of tyrosine within the protein to be measured. The standard protein must contain approximately the same number of tyrosine residues as the sample, or the procedure will be inaccurate. If there are no tyrosine residues in the sample to be measured, the Lowry method of protein determination is useless.

Lowry assay
Although there are many different proteins in any given cell, they all have one thing in common - the peptide bond which links the different amino acids together. Most protein assays actually test for the presence of peptide bonds. Every protein has, on average, the same number of peptide bonds per unit weight. The Lowry assay involves two reactions:
a) the reaction of peptide bonds with alkaline copper and
b) the reduction of phosphomolybdate by tyrosine and tryptophan residues.
From the experiment that we had conducted, we record the data in table and from the data, we sketch the graph of it. Below are the data and the graph from that experiment:

The result was not accurate since the graph for absorbance vs. concentration should have straight ascending graph.

There are several factors that might be the reason for the inaccuracy of our result. The factors are:
i. The Lowry method involves two redox reactions. It is more sensitive than the biuret assay; however, the Lowry assay is affected by interference from many common laboratory reagents and chemicals. Lowry assay may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents. Thus, it gave inaccurate readings of absorbance since it is so sensitive.
ii. The Lowry and copper/bicinchoninic assays are based on reduction of Cu2+ to Cu1+ by amides. Although this makes them potentially quite accurate, they require the preparation of several reagent solutions, which must be carefully measured and mixed during the assay. There might be some mistakes in measuring the lowry assay solution that might interfere the result.
iii. This is followed by lengthy, precisely timed incubations at closely controlled, elevated temperatures, and then immediate absorbance measurements of the unstable solutions. During the experiment, we only use our watch to count the time. Maybe there is some missed calculation in setting the time.

For the theoretical result, it should have an ascending straight line graph as shown below. Theoretically, the higher the concentration of the solution, the higher is the absorbance. Thus, it should give us straight ascending line graph as below.

We are also been given the samples of albumin from Grade A, Grade B and Grade C chicken egg and also the albumin of the duck egg. We need to determine the concentration of the egg. Below is the data that we collect through the experiment:

From the graph 1, we can conclude that the greater the absorbance the greater the concentration on protein contain in it. Thus, from our result, we conclude that grade A chicken A have greater concentration on protein, followed by Grade B chicken egg and lastly Grade C chicken egg. But, the duck egg (X) contain more concentration of protein compared to chicken egg. The concentration of the protein in egg is determine by the albumin that contain in egg. Albumin also known as egg white. Albumen accounts for most of an egg's liquid weight, about 67%. It contains more than half the egg's total protein, niacin, riboflavin, chlorine, magnesium, potassium, sodium and sulfur. Thus, the bigger the egg size, the greater it contain albumin that is a main sources of protein in egg.

The protein in eggs has a biological quality greater than any other natural food. In fact many manufacturers of protein powders often base their products on egg protein such as albumin because of its protein quality. Egg protein contains all the essential amino acids in the exact proportions required by the body for optimum growth and maintenance of lean, metabolically active tissue.