Protein Analysis - Nonenzymatic Protein Function and Protein Analysis - MCAT Biochemistry Review

MCAT Biochemistry Review

Chapter 3: Nonenzymatic Protein Function and Protein Analysis

3.4 Protein Analysis

Separating proteins from one another is generally only the first step in analysis. The next step is to study the isolated protein. Protein structure, function, or quantity is often of interest for a researcher or a commercial laboratory. Even after protein identification, protein analysis tools may be used. For example, in the case of protein synthesis for commercial use, purity of the product must be periodically assessed. The protein can be studied as a whole or broken down so that its parts can be examined.


Protein structure can be determined through X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Before crystallographic analysis, the protein must be isolated and crystallized. X-ray crystallography is the most reliable and common method; 75 percent of the protein structures known today were analyzed through this method. Crystallography measures electron density on an extremely high-resolution scale and can also be used for nucleic acids. An X-ray diffraction pattern is generated in this method, as shown in Figure 3.9. The small dots in the diffraction pattern can then be interpreted to determine the protein's structure.

Figure 3.9. Diffraction Pattern in X-Ray Crystallography

The minority of protein structure determination (25 percent) has been accomplished through NMR, as discussed in Chapter 11 of MCAT Organic Chemistry Review.


The amino acids that compose a protein can be determined by complete protein hydrolysis and subsequent chromatographic analysis. However, the random nature of hydrolysis prevents amino acid sequencing. To determine the primary structure of a protein, sequential digestion of the protein with specific cleavage enzymes is used. Small proteins are best analyzed with the Edman degradation, which uses cleavage to sequence proteins of up to 50 to 70 amino acids. The Edman degradation selectively and sequentially removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.

For larger proteins, digestion with chymotrypsin, trypsin, and cyanogen bromide, a synthetic reagent, may be used. This digestion selectively cleaves proteins at specific amino acid residues, creating smaller fragments which can then be analyzed by electrophoresis or the Edman degradation. Because disulfide bridges are broken to reduce the protein to its primary structure, their positions cannot be determined by these methods.


Like PCR gene sequencing, protein amino acid sequencing can be automated in a stepwise manner. By combining the information from both techniques, researchers can determine where on a chromosome the gene coding a particular protein resides.


Protein activity is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard. Activity is correlated with concentration but is also affected by the purification methods used and the conditions of the assay. Reactions with a color change have particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest.


Concentration is determined almost exclusively through spectroscopy. Because proteins contain aromatic side chains, they can be analyzed with UV spectroscopy without any treatment; however, this type of analysis is particularly sensitive to sample contaminants. Proteins also cause colorimetric changes with particular reactions, particularly the bicinchoninic acid (BCA) assay, Lowry reagent assay, and Bradford protein assay. The Bradford method is most common because of its reliability and simplicity in basic analyses.

Bradford Protein Assay

The Bradford protein assay mixes a protein in solution with Coomassie Brilliant Blue dye. In its protonated form, this dye exists as a brown-green color, as seen in Figure 3.10. The dye is deprotonated by the protein and gives up protons to the ionizable groups in the protein, turning blue in the process. Noncovalent attractions between the deprotonated dye and the protein then stabilize this blue form of the dye; thus, increased protein concentrations correspond to a larger concentration of blue dye in solution. Samples of known protein concentrations are reacted with the Bradford reagent and then absorbance is measured to create a standard curve. The unknown sample is then exposed to the same conditions, and the concentration is determined based on the standard curve. This is a very accurate method when only one type of protein is present in solution, but because of variable binding of the Coomassie dye with different amino acids, it is less accurate when more than one protein is present. The Bradford protein assay is limited by the presence of detergent in the sample or by excessive buffer.

Figure 3.10. Bradford Protein Assay The acidic form (left) has a brown-green hue; the basic form (right), which is created by interactions with proteins in solution, has a brilliant blue hue.

MCAT Concept Check 3.4:

Before you move on, assess your understanding of the material with these questions.

1. Why are proteins analyzed after isolation?

2. What factors would cause an activity assay to display lower activity than expected after concentration determination?

3. True or False: The Edman degradation proceeds from the carboxy (C-) terminus.