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# Amino Acids: Isoelectric Point?

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Hi! I'm just a little confused with respect to finding the pI of an amino acid with multiple charged groups.

Which is correct?

1) The pI = the average of all the pKa values of each group (pka1+pka2+pka3/3)

or

2) The pI = the average of the pKa values on either side of the zwitterion?

If you could provide an example with the explanation I would REALLY appreciate it! Thanks!

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Hi! I'm just a little confused with respect to finding the pI of an amino acid with multiple charged groups.

Which is correct?

1) The pI = the average of all the pKa values of each group (pka1+pka2+pka3/3)

or

2) The pI = the average of the pKa values on either side of the zwitterion?

If you could provide an example with the explanation I would REALLY appreciate it! Thanks!

Firstly, pI is pKa value where the net charge on the amino acid is zero (zwitterion). For a non polar amino acid (for example alanine), there are two reactive groups (the amino group and the carboxyl group). The pKa for the carboxyl is group is ~ 2 and for the amino group it is ~9. So if you think about it, the formation of zwitterion depends on the pH of the environment the amino acid is in. At pH 1 (a really acidic conditions) H bonds do not want to dissociate from the carboxyl group let alone the amino group, so you will have COOH and NH3+. The net charge is +1 on the amino acid here. As you increase the pH say to 5, the H in COOH group dissociates while the amino group still has the H ( COO- and NH3+) <--- this has a net charge of zero and is known as a zwitterion. To find the pI of non polar aa you need to find the average pKa values of the two reactive groups (2 + 9/ 2 = ~5.5). This pI value will be the same for all non polar aa.

This is made a little complicated with polar amino acids. Just know that for acidic aa the pI is reached when the H bond in COOH dissociates while the acidic R group retains its H bond and the amino group still has its H and thus has a positive charge on it (NH3+). To find the pI for an acidic aa, find the average of the pKa of the carboxyl group and the pka of the r group. In the case of glutamic acid, it will be pKa 2 for carboxyl group, pka of 4 for r group, and 9 for amino group. The pI will be 2 + 4/ 2 = 3.

Exact same principle for basic aa. The only difference is that to the find the pI, average the pKa of the r group and the amino group. With lysine for example, pka for carboxyl group is 2, pka of r group is 10, and pka of 9 for the amino group. The pI will be 10 + 9/ 2 = 9.5

Hope that helps!

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Firstly, pI is pKa value where the net charge on the amino acid is zero (zwitterion). For a non polar amino acid (for example alanine), there are two reactive groups (the amino group and the carboxyl group). The pKa for the carboxyl is group is ~ 2 and for the amino group it is ~9. So if you think about it, the formation of zwitterion depends on the pH of the environment the amino acid is in. At pH 1 (a really acidic conditions) H bonds do not want to dissociate from the carboxyl group let alone the amino group, so you will have COOH and NH3+. The net charge is +1 on the amino acid here. As you increase the pH say to 5, the H in COOH group dissociates while the amino group still has the H ( COO- and NH3+) <--- this has a net charge of zero and is known as a zwitterion. To find the pI of non polar aa you need to find the average pKa values of the two reactive groups (2 + 9/ 2 = ~5.5). This pI value will be the same for all non polar aa.

This is made a little complicated with polar amino acids. Just know that for acidic aa the pI is reached when the H bond in COOH dissociates while the acidic R group retains its H bond and the amino group still has its H and thus has a positive charge on it (NH3+). To find the pI for an acidic aa, find the average of the pKa of the carboxyl group and the pka of the r group. In the case of glutamic acid, it will be pKa 2 for carboxyl group, pka of 4 for r group, and 9 for amino group. The pI will be 2 + 4/ 2 = 3.

Exact same principle for basic aa. The only difference is that to the find the pI, average the pKa of the r group and the amino group. With lysine for example, pka for carboxyl group is 2, pka of r group is 10, and pka of 9 for the amino group. The pI will be 10 + 9/ 2 = 9.5

Hope that helps!

Thank you SO much for this!! Out of all the info I read, this made sense! Just a question about the non polar aa you talked about in the beginning..."As you increase the pH say to 5, the H in COOH group dissociates"...does the H in the COOH group dissociate at a ph above 2 and below 7? (and below 2 and above 7, it would not dissociate?)

Thanks again!

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Thank you SO much for this!! Out of all the info I read, this made sense! Just a question about the non polar aa you talked about in the beginning..."As you increase the pH say to 5, the H in COOH group dissociates"...does the H in the COOH group dissociate at a ph above 2 and below 7? (and below 2 and above 7, it would not dissociate?)

Thanks again!

The COOH will dissociate at a pH above 2. Once its dissociated, it's dissociated. So any pH above 2 COOH will become COO-. The amino group will dissociate above pH 9. Any pH below 9 the amino group will be positive (NH3+) and any pH above 9 it will dissociate to be come NH2. At pH of 11, for example, you have both groups dissociated as the COOH dissociated at 2 and NH3+ at 9, so it becomes COO - and NH2 (net negative charge)!

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The COOH will dissociate at a pH above 2. Once its dissociated, it's dissociated. So any pH above 2 COOH will become COO-. The amino group will dissociate above pH 9. Any pH below 9 the amino group will be positive (NH3+) and any pH above 9 it will dissociate to be come NH2. At pH of 11, for example, you have both groups dissociated as the COOH dissociated at 2 and NH3+ at 9, so it becomes COO - and NH2 (net negative charge)!

Thank you ! So for example, if I have Lysine at pH 11, the COOH group will be COO-, the NH3 will be deprotonated to NH2 and the lysine R group will make the pI will be 9.5. What would the charge be on the ion?

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pI = average of the 2 closest pKa values (if there's >2 charged functional groups)

you should do yourself a favour and figure out why that is though

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Thank you ! So for example, if I have Lysine at pH 11, the COOH group will be COO-, the NH3 will be deprotonated to NH2 and the lysine R group will make the pI will be 9.5. What would the charge be on the ion?

pI is when the net charge is 0...so if the COOH is COO- and NH3+ is NH2 at 9.5, then your r group would need to have a positive charge on it to cancel out the COO-

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