Characterization of Amino acids and Proteins can be done using different techniques utilizing the information based on isoelectric point and net charge.
I) Characterization of Amino acids and Proteins-Separation based on charge or pI
A) Electrophoresis:
One of the best method for the characterization of amino acids and proteins. In this method, an electric field is applied across a solid support (polymer gel, starch, paper). The solid support is saturated with buffer/protein solution. Depending on the charge of the protein it will move towards either the cathode (-)or the anode (+) or remain stationary (if pH=pI).
Therefore one can say that in electrophoresis, an electric current is used to separate a mixture of amino acids; the positively charged amino acids (pH < pI) move toward the negative electrode, the negatively charged amino acids (pH > pI) move toward the positive electrode an amino acid at its pI does not migrate; the amino acids are identified as separate bands on the filter paper or thin layer plate.
Example 1: (characterization of amino acids and proteins)
Consider a mixture of the amino acids lysine, valine, and aspartic acid at pH 6.0 that is subjected to an electric voltage. Which is which?
Solution:
pI value of lysine 9.7, which means pH < pI, therefore lysine will be positively charge and will move towards cathode
pI value of aspartic acid 2.8, which means pH > pI, therefore aspartic acid will be negatively charge and will move towards anode. pI value of valine 6.0pH, which means pH = pI, therefore aspartic acid will remain stationary.
B) Isoelectric Focusing- Electrophoresis Technique (characterization of amino acids and proteins)
The other valuable method for the characterization of amino acids and proteins is IEF. IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels. IPGs are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines. An Immobiline is a weak acid or base defined by its pK value.
A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode (negatively charged electrode). As it migrates through a gradient of increasing pH, however, the protein’s overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction towards either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.
Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a “positive” anode and “negative” cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the “positive” end while positively charged molecules move toward the “negative” end. As a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. At this point the molecule no longer has a net electric charge and as such will not proceed any further within the gel.
Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01.Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE.
So, isolectric points and net charge of amino acids can be well utilized in characterization of amino acids and proteins
C) Ion Exchange Chromatography (characterization of amino acids and proteins)
Ion exchange chromatography is well use for characterization of amino acids and proteins as it separates proteins or other molecules based on differences in their accessible surface charges. In ion exchange chromatography the analyte molecules are retained on the column based on coulombic (ionic) interactions. The stationary phase surface contains ionic functional groups of opposite charge that interact with analyte ions. The elution is done by increasing salt gradient. Most commonly used salt is NaCl, exists in equilibrium with Na+ (cation) and Cl- (anion) in aqueous solution. As the concentration of salt increases concentration of Na+ (cation) and Cl– (anion) also increases. The basic principle of ion exchange chromatography is the reversible exchange of analyte ions bound to solid support with similar ions generated from salt in liquid phase.
Many biological molecules such as proteins, amino acids, nucleotides and other ions have ionisable groups which carries a net charge (positive or negative) dependent on their pKa and on the pH of the solution, which can be utilized in separating mixture of such. Ion exchange chromatography experiments are carried out mainly in columns packed with ion exchangers. On the basis of type of exchanger used for separation this chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography.
During characterization of amino acids and proteins if one uses Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group cations. Anion exchange chromatography retains anions using positively charged functional group.
Many biological molecules, especially proteins, are stable within a narrow pH range so the type of exchanger selected must operate within this range for the better characterization of amino acids and proteins.
Suppose if protein is most stable below its isoelectric point (pI), there will be net positive charge on the protein surface, so for separation of this protein cation exchanger should be used (experimental pH value should be between lowest pH where protein is stable). Commonly used cation exchange resins functional groups are following: Carboxymethyl (CM), Sulphopropyl (SP), Methyl sulphonate (S)
If protein is most stable above its pI, there will be net negative charge on the protein surface and anion exchanger should be used (experimental pH value should be between highest pH where protein is stable and pI value). Commonly used anion exchange resins functional groups are following, Aminoethyl (AE-), Diethylaminoethyl (DEAE-), Quaternary aminoethyl (QAE-).
If protein is stable over a wide range of pH, it can be separated by either type of ion exchanger (experimental pH value may be decided considering lowest and highest pH value stability of the protein). Weak electrolyte requires very high or very low pH for ionisation so it can only be separated on strong exchanger, as they only operate over a wide pH range, whereas in case of strong electrolytes, weak exchangers are preferred.
So these are some methods used for the characterization of amino acids and proteins but there are now more well advanced techniques-Mass spectrometry, Circular Dichroism Spectrometry, western blotting etc.,
