Determining Protein Concentrations of Unknown Solutions: The Biuret Protein Assay – Biology Research Paper
This report details the use of the Biuret Method of protein assay to determine the protein concentrations of two unknown solutions. To determine the protein concentrations of the unknowns, six solutions with known protein concentrations were passed through the spectrophotometer and their absorption levels discovered. Using the protein concentrations and absorption levels to plot a
standard curve, and determining the absorption levels of the unknown solutions (which were later revealed to be Ovalbumen and Milwaukee’s Best brand beer), it was then possible to determine their protein concentrations. Analysis of the data revealed that in the solutions containing more than .2ml of water, levels of protein concentration and absorption were closely related.
Why should you care about protein concentrations in solutions? There are a number of reasons. In many lab experiments, for example, determining the amount of protein biomolecules in a given solution is essential. One objective of this lab was to learn a method by which protein concentrations could be determined in unknown solutions. In addition, the experiment was performed to determine if absorbance levels correspond with protein concentrations in solutions.
In addition, it was important that the method by which the protein concentrations of various substances were determined apply universally. In other words, the method must work in every test in which it is used. The method chosen for this experiment was the Biuret Assay method, which requires that the solutions being tested be mixed with a Biuret reagent and run through a spectrophotometer (“Brad Olson’s Protein Assay Resource”).
For this experiment, six vials were each filled with 4.0ml of Biuret reagent, varying amounts of Ovalbumen (obtained from egg whites) (in an evenly staggered progression of 0.0-1ml) and water (arranged in an evenly descending order from 1-0ml). The protein concentration of the solutions in these vials corresponded directly with the amount of Ovalbumen in the vial (e.g., .2ml Ovalbumen yielded a 2mg/ml protein concentration). With this knowledge, the x-axis of a graph was divided into ten equal sections (0-10mg/ml). To determine the absorbance of these solutions, 3 ml of each solution was placed in a previously zeroed spectrophotometer, and readings were taking, ranging from an absorbance of 0 to .654. Using this information, the y-axis of the graph was divided equally from 0-.9. Using the data to plot six points and drawing a standard curve through them, the next step was to then determine the absorbance of two unknown solutions, chosen from four, labeled A-D. Mixing 1ml of the unknowns with 4ml of Biuret for each sample, they were run through the spectrophotometer, and the absorption was successfully determined. Using the absorption data as a guide, the protein concentration was estimated and graphed.
For the first four Ovalbumen (control) samples, the protein concentration lined up with the level of absorbance, provided that the amount of water in the solution exceeded .2ml. (see fig. 1). Take, for example, the solution which contained .6ml water and .4ml Ovalbumen. With a protein concentration of 4mg/ml, the readings from the spectrophotometer calculated the solution’s absorbance at .397 (see fig. 1).
Using the data from the six samples, a standard curve was graphed. The remaining two samples, (A and D) both unknown solutions, were run through the spectrophotometer, and had absorption levels of .106 and.112, respectively. Finding these points on the y-axis, a line was then drawn to the curve, and the intersection of these points were estimated, providing protein concentrations of .78mg/ml for sample A (Ovalbumen) and .79mg/ml for sample D (Milwaukee’s Best brand beer) (see fig. 1) For the sake of accuracy, each solution contained no water, 4ml of the Biuret reagent, and 1ml of the sample substance.
Once the amount of water dropped below .4ml, however, there were adverse effects on the absorbance level of the solution, as evidenced by the data taken from the final two Ovalbumen-based solutions. Containing .2 and 0 ml of water, their absorbance remained around .6, despite their protein concentrations of 8 and 10mg/ml, respectively (see table). These results were initially attributed to human error, but after ensuring accuracy of measurement in a second test, the spectrophotometer delivered similar numbers. The conclusions was made that the low levels of water in the last two samples (.2 and 0ml) contributed to the results.
Because the data of this experiment was used to create a standard curve graph for absorption/protein concentration of known solutions, it is possible to accurately identify protein concentrations in any number of unknown solutions. The implications of this are far-reaching, with applications in many fields, including (but not limited to) nutritional and food science. One implication of these results is that protein concentration and absorbance are linked, and correspond to each other, as was initially predicted (see fig. 1). Also, it is important to note that this link is dependent on the amount of water in a solution. As the amount of water in a solution drops, the absorption level of a solution increases, as does protein concentration. By using this data to graph a standard curve, it is not only possible, but easy to determine the protein concentration of any substance by first determining its level of absorption.
Olson, Brad. Brad Olson’s Protein Assay Resource 2000. University of Nebraska-Lincoln Dept. of Biochemistry. 13 September 2005. < http://www-class.unl.edu/biochem/protein_assay/>.