How to Calculate Isoelectric point

The specific pH at which the net electric charge of an amino acid is zero, known as isoelectric point or isoelectric pH, denoted as pI. Every amino acid has its own specific pI value. Every amino acid has inonizable groups i.e., COOH and NH₂. If amino acid is acidic viz. Aspartic acid and Glutamic acid it has extra COOH in R chain whereas, in case of basic amino acid viz. Arginine and Lysine, one extra NH₂ group is found in its R chain.

⁺H₃N-CH(R)-COOH           →      ⁺H₃N-CH(R)-COO^–          →         H₂N-CH(R)-COO^–

^{Net charge        +1                                               0                                                   –1}

When these ionizable groups ionize give characteristic pKa values. When amino acids having nonionizable R groups give only two pKa values i.e., pKa₁ & pKa₂ and amino acids with nonionizable R groups give three pKa values i.e., pKa₁(–COOH), pKa₂(–NH₂) and pKa_R(–COOH or –NH₂ of R chain)

These pKa values are very important in calucaltion of isoelectric point (pI).

Calculation of pI:

One can calculate pI, easily by following formula

(1)   Amino acid with nonionizable R groups

pI = (pKa₁ + pKa₂)/2

Example: pI of Alanine (pKa₁= 2.34, pKa₂= 9.69)

pI = (2.34 + 9.69)/2 = 6.01

(2)   Negatively charged (acidic) amino acid

pI = (pKa₁ + pKa_R)/2

(pKa_R = pKa value of extra –COOH in R chain)

Example: pI of Aspartic acid (pKa₁= 1.88, pKa₂= 9.60, pKa_R= 3.65 )

pI = (1.88 + 3.65)/2= 2.77

(3)   Positively charged (basic) amino acid

pI = (pKa₂ + pKa_R)/2

            (pKa_R = pKa value of extra –NH₂ in R chain)

Example: pI of Arginine (pKa₁= 2.17, pKa₂= 9.04, pKa_R= 12.48)

pI = (9.04 + 12.48)/2= 10.76

Isoelctric point (pI) of 20 standard amino acids:

Amino acid	pK1	pK2	pKR	pI
Glycine	2.34	9.60		5.97
Alanine	2.34	9.69		6.01
Proline	1.99	10.96		6.48
Valine	2.32	9.62		5.97
Leucine	2.36	9.60		5.98
Isoleucine	2.36	9.68		6.02
Methionine	2.28	9.21		5.74
Phenylalanine	1.83	9.13		5.48
Tyrosine	2.20	9.11	10.07	5.66
Tryptophan	2.38	9.39		5.89
Serine	2.21	9.15		5.68
Threonine	2.11	9. 62		5.87
Cysteine	1.96	10.28	8.18	5.07
Asparagine	2.02	8.80		5.41
Glutamine	2.17	9.13		5.65
Lysine	2.18	8.95	10.53	9.74
Arginine	1.82	9. 17	12.48	10.76
Histidine	2.17	9.04	6.00	7.59
Aspartatic acid	1.88	9.60	3.65	2.77
Glutamic acid	2.19	9. 67	4.25	3.22