Structural Biochemistry/Quantifying Proteins
Knowing the quantity of a protein after each separation step is useful in checking the progress of purification and evaluating the technique's efficiency. Quantifying proteins also helps us understand how an organism functions as one. Several chromatography techniques rely on quantifying proteins by mass, with additional observables such as charge to provide further differentiation.
Because specific activity is a ratio of the enzymatic reactions of a particular protein to the total amount of proteins, quantifying a protein can be followed throughout a purification. The equation for specific activity can be modeled as: . Therefore, as the total amount of protein decreases per step, the specific activity should rise. Generally, an assay performed will give the rate of reaction, in units such as micromoles per second. Dividing this rate by the concentration of your enzyme preparation yields the specific activity of a protein.
Ideally, the end of purification should be consistent with a constant specific activity. The specific activity can be monitored and used to quantify a purification by analyzing several variables which are total protein, total activity, yield, and purification level.
The concentration of a protein can be measured by immunological techniques such as ELISA or Western Blotting (the former being able to measure the quantity of protein present because of the direct proportionalities of reagents to proteins).
Activity can be measured using fluorescent techniques.
In order to determine how much activity is retained after each successive purification step in the crude extract, the yield can be calculated as . In order to convert this to a percentage, multiply the yield by (100). Also, it is important to note that in most cases, the amount of initial activity is always 100 %.
By obtaining a value for the purification level, we are able to assess how much purity has increased. The purification level can be calculated by: (
- Important note: a purification scheme turns is only successful when taking into account BOTH purification levels and percent yield. Experimentation can become fairly complex if there is a high yield with very little purification. This is because there is an indication that there are a vast number of contaminants/proteins that aren't of interest. On the other hand, a purification level is high while the percent yield is low, then it is fair to conclude that there isn't enough protein available to carry out the experiment.
Total Number of ProteinsEdit
The amount of protein separated using chromatography or dialysis is determined by:
Total Enzymatic ActivityEdit
The recovered volume's activity is determined by:
In addition to electrophoresis and immunological assays, the use of ammonium sulfate, (NH4)2SO4, can also quantitatively evaluate a purification. Because ammonium sulfate is non-denaturing and very water soluble (it is high on the Hofmeister series), it is used to effectively precipitate proteins: at high concentration, the ammonium and sulfate ions absorb most of the water through hydroelectric attraction, leaving the proteins to aggregate and precipitate out. ^
The mass of a protein can be measured using the sedimentation-equilibrium technique. This method requires slow centrifugation of a sample in order to establish a balance between sedimentation and diffusion. Unlike SDS-Polyacrylamide Gel Electrophoresis, which gives merely an estimate of the mass of dissociated and denatured polypeptide chains, sedimentation-equilibrium provides accurate mass measurements without requiring denaturation, thereby allowing the native structure of multimeric proteins to be left intact. Furthermore, the number of copies of each polypeptide chain that are present in a multimeric protein can be determined based on the mass of the dissociated chains and the mass of the entire multimeric protein, as measured by SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium, respectively.
Mass spectrometry is another accurate analytical technique for determining protein mass. In this technique, atoms are ionized through a machine and passed through a vacuum into the detector. In which then, the time of flight (TOF) in the electrical field is directly proportional to the mass of the protein (or the mass-to-charge ratio). Thus, the smallest protein in a protein mixture has the smallest TOF, whereas the largest protein has the largest TOF. This technique allows the identification and analyzation of molecules based on their size and mass. This approach however, does not entail too much information about the structure or conformation of a protein.
-  "Chapter 9: Protein expression, purification and characterization", Proteins: Structure and Function, Whitford, 2005, John Wiley & Sons, Ltd
Biochemistry, 6th ed., Berg et al., 2007 Freeman