Report On Protein Quantification Using Two Independent Spectrographic Techniques

Introduction

Accurately measuring the protein concentration is necessary in biochemistry as the proteins are used for measuring the activity in the case for example enzyme reactions (Aitken, 1996). Usually determining the protein concentration is the first step in a biochemical protocol and have inaccuracy can lead to overall inaccuracies in all the experiments. However, getting an accurate protein concentration is daunting and although there has been nearly 100 years of biochemical research on proteins no method yields absolutely accurate results. However, direct spectroscopy (if the extinction coefficient is known and Lowry technique are good methods and offer good sensitivity (Aitken, 1996). With direct spectroscopy you use the Beer-Lambert law and either calculate the concentration from the absorbance with a calculated or known molar extinction coefficient experimentally (Olson, 2007).
In this experiment we will be using the Biuret and Lowry methods for accurate determination of the protein. In both we will detect using an absorbance spectrometer at different wavelengths. This law shows that the concentration is proportional to absorbance over the attenuator (in this case protein, water and copper II solution) and pathlength of the cuvette. The Biuret method utilizes copper II in an alkaline solution and is in excess to the protein. It measures protein concentration from 5-160 mg/mL. Upon addition of peptide it forms a violet colored coordination complex in solution. With increasing concentration the same frequency of peptide bonds occur increasing the intensity of colour. At a wavelength of 540 nm the intensity of color the protein concentration can be measured (Olson, 2007). The Lowry technique can detect much lower concentrations of protein (0.1 - 1 mg/ml) and relies on two reactions. The first copper ion complex with amide to produce a Biuret chromophore. The second is not quite understood but involves the reduction of the Folin-Ciocalteu reagent with aromatic residues. The reaction allows us to visualize from 500- 750 nm. The Folin-Ciocalteu reaction allow a higher sensitivity. Most researchers use 660 nm or 750 nm depending on whether there is potential contamination e.g. chlorophyll (Lowry, 1951; Olson, 2007). Our aims of this experiment is to quickly and accurately quantify the protein using these methods.

Method

We had available to us 10 tubes, Biuret and Lowry reagents, an absorbance spectrometer. Known concentrations of Bovine Serum Albumin (BSA) was provided. Unknown concentrations of gelatin and gamma globulin were given. A calibration curve was made with both the Biuret method and Lowry method using bovine serum albumin. For the Biuret method 0, 1, 2, 3, 4, 5 mg/ml concentrations of the albumin was used and Biuret solution was added, mixed and left for 15 minutes. The tubes were placed in the absorbance spectrometer set to 540 nm and the absorbance was recorded. For the Lowry method bovine serum albumin concentrations of 0, 20, 40, 60, 80, 100 ug/ml were used and Biuret reagent was added, mixed and left for 15 minutes followed by Folin-Ciocalteu reagent which was added, mixed and left for 20 minutes. Tubes were placed in the absorbance spectrometer set to 700 nm and the absorbance was recorded against blank. Unknown amounts of gamma-globulin and gelatin were given and these were tested in duplicate on both calibration curves. Results were recorded and the graph was used to get the final concentration of each protein.

Results

Discussion
We obtained good satisfactory results for both methods. In our protein assays we determined the unknown concentrations by comparing the absorbance to the absorbance of a dilutions series of known standards concentrations in this case Bovine Serum Albumin. Both methods provide a high level of accuracy although . Several sources of error could be found in this method the Lowry method is more sensitive and can go to 10 times lower concentration (Olson, 2007). These could be several chemicals which can interfere with this assay in particular: glycerol, detergents, carbohydrates, Tricine, EDTA, Tris, disulfide compounds, potassium compounds, sulfhydryl compounds, magnesium and calcium. Most of these interfering substances are commonly used in buffers for preparing proteins (Olson, 2007). As well all the glassware should be washed to avoid any interference in the cell. Secondly by keeping the reaction going too long the color intensifies. So if we did not do these in series then we might have obtained vastly different results. The Lowry method is understood by its reactivity with particular residues e.g. cysteines, asparagine, histidine and aromatic amino acids. For two similar proteins that have different levels of aromatic amino acids the concentration might be slight different if using the Lowry technique (Lowry 1951). It is known that the Folin-Colinteau solution may react contaminants like urea so this technique may not be helpful if there is urea like to measure protein in urine samples. In our case we did not seem to have contaminates so it could be measured at 700 nm. In general the Lowry method would be a better method to measure the protein concentration of an unknown sample as it is more sensitive.
Generally with these kind of experiments you want the UV instrument to be on for a little while before starting. We relied on the technicians to make sure that the spectrophotometer is calibrated correctly. If there is something wrong with the light path, the bulb or the grading there might be significant changes in the standard curves. The blank also needs to be zeroed to make sure that the material inside is not causing any wrong readings. If all these are correct we can have confidence that our final results are correct (Olsen, 2007).

References

Gornall, Allan G., Charles J. Bardawill, and Maxima M. David. (1949) Determination of serum proteins by means of the Biuret Reaction. Journal of Biological Chemistry 177.2 : 751-766.
Aitken, Alastair, and Michèle Learmonth. Protein determination by UV absorption.(1996) The protein protocols handbook. Humana Press,. 3-6.
Olson, Bradley JSC, and John Markwell.(2007) Assays for determination of protein concentration.Current protocols in protein science : 3-4.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1): 265–75.
Gill, Stanley C., and Peter H. Von Hippel (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Bioch.182.2 : 319-326.