A very simple yet confusing topic regarding the CSIR net examination is isoelectric point which is mainly used in the isoelectric focusing technique to predict the amino acid Composition of a protein and the range of the buffer required for the electroforesis I hope this topic will help you to solve these questions at a galnce. so please start this topic by having a brief description about titration curve- acid base titration involves the gradual addition or removal of protons. at very low pH the predominant ionic species of glycine is +H3N-CH2-COOH, The fully protonated form at the midpoint in the first stage of the titration, in which - COOH Group of the glycine loses it’s proton, equal molar concentrations of the proton Donor (+H3N-CH2-COOH) and acceptor (+H3N-CH2-COO-) Species are present. at the midpoint of any titration, a point of inflection is reached where the pH is equal to the pKa (It is the range of PH at which any group donate its proton ) of the protonated group being titrated. for glycine the pH at the midpoint is 2.34 thus it's -COOH group has a pKa of 2.34. As the titration process proceeds another important point is reached at PH 5.97. there is another point of inflection at which removal of the first Proton is essentially complete and the removal of the second has just begin. the pk for the NH3+ group of glycine is 9.60. the carboxyl group of the glycine is hundred times more acidic than the carboxyl group of Acetic Acid. the pKa of the glycine’s -COOH Is lower due to the repulsion between the positively charged NH3+ group, while the pKa for the NH3+ is much higher due to the attraction between the positive Ion of NH3+ and COO- , The second piece of information from the titration curve can be achieved that the range of PH where the buffer of particular compound is effective. Eg;- The glycine has the pKa about 2.34 for the -COOH so it is a good buffer to maintain the pH 2.34 and it has pKa about 9.60 for NH3+ so it is a good buffer for near 9.60 pH range so glycine is not a good buffer to maintain the physiological PH near 7.4.
The henderson-Hasselbaich equation (pH = pKa+log(proton acceptor/proton donor) can be used to calculate the proton donor and proton acceptor ratio to make a buffer to maintain a certain pH.
Titration curves predict the electric charge of amino acids:- The titration curve of an amino acid predicts the relationship between it's net electric charge and pH of the solution . The characterstic pH at which the net electric charge is zero called the Isoelectric point or Isoelectric pH. For glycine which has no ionizable group in it's side chain the isoelectric point is simply the mean of the two pKa values-
PI (Isoelectric point)= (pK1+pK2)/2
so for glycine the Isoelectric point will be = 2.34+9.60/2= 5.94
The important points which we get after reading the above given information is that At higher pH than the PI of the glycine it will contain negative charge and will move towards the anode(+).
At pH less than PI, It will possess positive charge and will move toward cathode(-).
Amino acids differ in their acid base tritation properties:- The glycine do not contain any ionizable R-group so it has only two pKa values, but some amino acids such as the glutamate,histidine contain the ionizable R-groups so they have three pKa values, additional pKa value for ionizable R-group. So they have pKa for pK1 (for -COOH), pKr (for R group) and pK2 ( for NH3+).
Calculation to predict the PI (Isoelectric point) for the amino acids having Ionizable R-group:-
The calculation for the isoelectric point of the amino acids having the acidic R-group such as Glutamate is
= pK1 (for -COOH)+pKr(for side chain group)/2
The amino acids having the R-group of basic nature such as histidine contain the imidazole ring , the PI will be
= pKr+pK2( for NH3+)/2
Important note:-
Only Histidine has the pKr near the physiological pH, pKr= 6.0. No other amino acid has pKa near 7 so histidine is found in the intracellular and intercellular fluids
in most animals and bacteria. The PI for histidine is 7.59.
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