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Protein studies using spectroscopy

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Introduction

Protein studies using spectroscopy Aisha Asam Biomedical sciences (HND) 27.11.03. Dr Caroline Dalton. Introduction The aim of the practical was to investigate two protein methods. Two spectroscopy methods were used. One of them was the Biuret method and the UV method. The Biuret method is based on the formation of coloured complexes, where the UV method depends on absorption of light in UV part of spectrum by certain amino acids in the protein's structure. In this experiment my aim was to find sample Y concentration. Biuret reaction- The Biuret reaction occurs upon treatment of peptides or proteins with an alkaline solution of cu2+ ions. As the copper ions are complexed by the amide nitrogen atoms of the peptide backbone, a violet colour is seen. Spectrophotometric determination of the amount of colour is used as an index of the quantity of peptide present. Various spectroscopic methods are employed in evaluating protein structure and function in ultraviolet light spectroscopy. Peptide and tyrosine bonds in proteins absorb UV light. The efficiency of light energy absorption for each chromophore is related to its molar extinction coefficient. ...read more.

Middle

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). ...read more.

Conclusion

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. ...read more.

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