Protein concentration assays: is a commonly used method for protein determination:
BCA Assay: in alkaline solutions, Cu2+ binds to peptide bonds of proteins. Cys, Trp, and Tyr, are capable of reducing the bound Cu2+ to Cu+ resulting in formation of a moderate purple colour proportional to the protein concentration. This colour is used in the Biuret assay to determine protein concentration. The sensitivity can be increased by addition of Bicinchoninic acid (BCA). When BCA binds Cu+ an intense purple colour proportional to the protein concentration is observed. However, because the content of reducing amino acids varies around other proteins, the colour yield per milligram of protein is not constant amongst different proteins.
Method
The standard solutions were made by dilutions of the standard proteins (figure 1 below)
Figure 1
Standard solutions were made from albumin with known concentration =20g/L as shown above.
The concentration was worked out by using:
The protein concentrations were from 0-20g. After the dilutions were mixed properly 0.5ml of every dilution was pipetted into test tubes which were labelled. At the same time five of the 0.5ml samples of Y were put into the test tubes. After getting each test tube prepared, Biuret reagent 4.5ml was added and mixed properly. Then for 15 minutes the solutes were left, to reach their maximum colour.
To analyse the samples the spectrometer was used and it was set to a wavelength of 542.7nm. A sample which contained 0ml albumin, 4.5ml of Biuret, 5ml distilled water was added to a cuvette, and spectrometer was set on 0.After setting on 0 the cuvette was taken out. The samples contained the standard solutions (water and Biuret) were analysed and the absorbance was recorded. After absorbance of Biuret was measured, the Y samples were looked at, under the same wavelength. A calibration graph was determined.
Five samples of Y were diluted this was done to determine the protein by UV absorbance (1 in 10 dilutions). The spectrometer was set to 278.7nm wavelength and then spectrometer was zeroed using distilled water (1cm). In a silica cuvette (silica cuvettes need to be rinsed before and after). From these results the other calibration graph was drawn. This method measured the absorbance of all the solutions.
RESULTS
I suggest these results are valid because the graph was as I expected.0.5ml albumin with added 4.5ml of biuret reagent.
The aim of the experiment was to find Y concentration, these results were successful.
At 278.7nm wavelength absorbance of UV method. For the diluted 1ml albumin in 9ml distilled water against the concentration. The graph I plotted using these results was valid, these results are also valid as they go up and a pattern/trend can be seen.
To find the concentration of Y. At 278.7nm the absorbance for the UV method for 1ml of sample Y in 9ml distilled water and the concentration was obtained from the graph.
These are my standard solutions, they are accurate/reliable.
Below is the table for my tyrosine results:
Standard Deviation -for the Biuret method -concentration of sample Y is below:
Standard deviation for the UV method -concentration of sample Y is below:
DISCUSSION
By Using the Biuret method I was able to find out the sample Y concentration. The colour changes depended on the amount of peptide bonds available in the protein. The colour was clear I would suggest the darker the colour was the higher the concentration and higher the absorbance.
With the UV method I was able to know the structure of the protein (eg if it contained amino acids). Because the absorbance is always proportional to the concentration. (The more aromatic amino acids present in the protein the more rise in protein).
Tyrosine and Alanine didn't affect the Biuret method, where as in the UV method it was clear that absorbance was different, this was because the tyrosine which is a phenol group absorbs more UV light which causes decolourisation of the electrons in the tyrosine's benzene ring so the absorbance was high. Where the alanine didn't absorb the UV light so the absorbance was small this is shown in my table of tyrosine results.
By looking at my standard deviation, for the biuret method. I would suggest it is very small (0.729) this means my results were reliable/verifiable and I didn't get any anomalous results. The standard deviation for UV method was (0.986). Something went wrong in my practical because the absorbance was low, this happened bacause I didn't mix the solution well or didn't zero the spectrometer. Below is how I identified there was something wrong with my results:
- CV was higher than 5% for biuret method (12.4%) and for the UV method (14.25%)
REFERENCES
- Biochemistry (by Garrett and Grisham)
- Principles of medical biochemistry (by Gerhard Meisenberg and William H.Simmons)
- Biochemistry (by Jeremy M. Berg and John L.Tymoczko) (5th edition)
- Principles and techniques of practical biochemistry (by Wilson and Walker) (5th edition.)
