Protein Structure - We have mentioned before that biological macromolecules are all polymers. Carbohydrates are sugar polymers. We built lipids from fatty acids, phosphate groups, and glycerol. Now we come to proteins, which are also polymeric. The subunits which make-up Proteins are Amino Acids. The amino acids are joined together by dehydration synthesis to form chains, which are hundreds of amino acids long; called proteins. Proteins function as enzymes or as structural units in cells. They do most of the "work" in a cell. Almost all of the exciting stuff; metabolism, memory, hormone action, and movement involves proteins. In this lab, we will learn one method of measuring protein concentration.

Procedures - The biuret reaction is a method that can be used to determine the amount of soluble protein in a solution. The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds (which join amino acids to form Proteins) and changes color when it does so. The spectrophotometer can then be used the measure the intensity of the color produced. The more protein present the darker the color.

Preparing a Standard Curve - In order to quantitavely determine how much protein is represented by a particular Absorbance reading it is necessary to construct a standard curve. This is done by performing the biuret reaction on a series of samples containing known amounts of protein. The Absorbance readings obtained from these samples are used to construct a graph of Absorbance as a function of protein concentration. This graph is called the standard curve for the assay, and can be used to convert the Absorbance readings for the experimental samples into a protein amount or concentration.

A new standard curve should be constructed each time you set up to run a series of protein determinations. The standard curve corrects for variations in experimental conditions (solution preparation, spectrophotometer sensitivity, temperature, etc.)

Protein Assay -
Before you start, zero your Spec 20. Since the Biuret solution components absorb some amount of light themselves, this amount must be accounted for by “subtracting” it from the total absorption for the protein alone. This is done by using the tube containing the Biuret solution only as a blank (this is like what you just did in the previous Spec 20 exercise so we will ignore it in our Lab preview).

To construct your standard curve, determine and record the absorbance values at 560 nm, successively, for the different protein concentrations. Using the graph area below, plot the data with Protein concentration on the horizontal axis and absorbance on the vertical axis.

(To review the Bio 121 Intro Lab graphing/standard curve material click here).

Build your standard curve - Click on the tubes below, which contain known protein concentrations in Biuret solution and determine their Absorbance.

Enter the absorbance values for the various protein solutions below. Then graph your data, add a "best-fit" line and answer the questions below. Print (or write down) and save your aborbance data, you will need them to construct a "standard curve". Protein Concentration Absorbance @ 560 nm 0 mg BSA/ml 250 mg BSA/ml 500 mg BSA/ml 750 mg BSA/ml 1500 mg BSA/ml Print out the graph below, You can then construct a practice standard curve using the above data. (click the graph to open in a new page for printing). Here is the Example Graph from the Intro Prelab if you haven't made standard curves before. Some Questions to answer for fun: 1. 560 nm wavelength corresponds to (approximately) what color in the spectrum?   2. In the Biuret reaction, which of the below protein solutions should have the highest absorbance @ 560 nm? 500 mg/ml 250 mg/ml 125 mg/ml 75 mg/ml On to the Unknowns