What is isoelectric point and how it is calculated?
The isoelectric point (pI) is the pH value at which the molecule carries no electrical charge. The concept is particularly important for zwitterionic molecules such as amino acids, peptides, and proteins. For an amino acid, the isoelectric point is the average of pKa values for the amine and the carboxyl group.
What is the formula to calculate isoelectric point?
These amino acids are characterised by two pKas : pKa1 and pKa2 for the carboxylic acid and the amine respectively. The isoelectronic point will be halfway between, or the average of, these two pKas, i.e. pI = 1/2 (pKa1 + pKa2).
What is meant by the isoelectric point of a protein?
The isoelectric or isoionic point of a protein is the pH at which a protein carries no net electrical charge and hence is considered neutral [1,2,3,4].
How can a protein’s isoelectric point be used in protein purification?
At the isoelectric point, a protein has no net charge. Above the isoelectric point, a protein carries a net negative charge—below it, a net positive charge. This convenient method thus purifies complex protein mixtures by helping proteins reach their respective isoelectric points.
What is the isoelectric point of amino acids?
The isoelectric point of an amino acid is the point at which the amino acid has no net electrical charge. It is an important characteristic for any amino acid, because every amino acid has at least two acid–base (titratable) groups.
How do you find the isoelectric point on a titration curve?
For a simple diprotic amino acid, the pI falls halfway between the two pK values. For acidic amino acids, the pI is given by ½(pK1 + pK2) and for basic amino acids it’s given by ½(pK2 + pK3).
How do you separate proteins by isoelectric point?
Separation of proteins at the isoelectric point is called isoelectric focusing. In isoelectric focusing a gradient of pH and an electric potential are applied across the gel, making one end more positive than the other. Separation occurs on the basis of the positive or negative groups present on the molecule.
How do you separate proteins based on its isoelectric point?
A protein can be purified according to its protein isoelectric point by running the said protein through an ion exchange column or a pH-graded gel. At the isoelectric point, a protein has no net charge. Above the isoelectric point, a protein carries a net negative charge—below it, a net positive charge.
Why do biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight?
The average molecular weight of the 20 standard amino acids is 138, but biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight. The number 110 reflects the higher proportion of small amino acids in proteins, as well as the loss of water when the peptide bond forms.