Introduction

This exercise will introduce the concept of using a color reaction to determine the protein content of an unknown solution. It will draw upon your familiarity with the use of the spectrophotometer and prepare you for future experiments in which you will be wanting to know the protein content of a solution which may contain other molecules which interfere with a simpler determination of protein content. It will also demonstrate and practice your abilities to make up accurate standards.
Theory
Protein absorbs light at the ultraviolet wavelength 280 nm by virtue of its relatively constant content of tyrosine and tryptophan residues. One can determine protein content of a pure protein solution by its absorbance at 280 nm; however if other uv absorbing compounds such as free tyrosine or uric acid are present in the solution, an erroneous estimate of protein concentration can be obtained. Numerous colorimetric assays specific for protein have been developed in the past. The most recent development in this respect uses a relatively colorless (actually a brownish orange) dye, Coomassie Brilliant Blue R-250, the leuco form of a molecule which turns an intense blue color on reacting with polypeptides (Sedmak & Grossberg, 1977, Analytical Biochemistry 79, 544-552). Such 'leuco' to 'colored' transformations are common in analytical biochemistry. This technique allows the use of a visible light colorimeter, such as the Spec-20/21s and Genesys V spectrometers, we have available in the laboratory, to measure protein solutions from a variety of sources in a rapid and extremely sensitive manner.

Technique
Making standard solutions: You will be supplied with a 1 milligram per ml solution of Bovine Serum Albumin. Dilute this solution with distilled water (or whatever physiological saline you are expecting your unknown to be in; a 0.15 N NaCl is 'safe' for most extracellular protein solutions, 0.15 N KCl for cellular.) to provide a linear range of 1.5 ml protein solutions ranging from 1 to 20 micrograms of protein per ml of solution. Make two independent dilution series for each standard curve you want. These independent dilutions will allow you to asses the errors involved in your technique.

The Assay: Equal volumes (0.75 ml) of the 'leuco' dye solution and the protein standard solutions are added together in ordinary glass 13x100 mm test tubes. The color develops almost immediately and should be read in the spectrophotometer within the next 90 minutes using the dye diluted (1:1) with distilled water (or saline as the situation requires) as a blank. Plot the resultant absorbance versus the protein concentration on graph paper to give a standard curve. Which should be X and which Y?

An unknown: Chose a biological fluid of unknown protein content (saliva, urine, human blood serum, tears, column fractions, etc.) and clear it of any particulate mater, if necessary, by centrifugation or filtration. Dilute it various orders of magnitude with appropriate solvent to be consistent with your standard curve in order to get its protein content into the linear range of your standard curve. This will have to be done by a trial and error process since you may have no prior information about the normal range of protein content in the unknown solution.

Evaluation: How consistently did your independent dilutions give you the same color reading. Are there any systematic departures from linearity along the length of standard curve? How would you go about determining if your unknown sample had a normal amount of protein in it? Comment on the accuracy vs precision of this colorimetric procedure.