I understand the method of how to determine the isoelectric point of a protein chain but I am confused with assigning the charges when looking at the different pka values. Can someone please explain this?
Isoelectric point of proteins
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First, a protein at its isoelectric point is neutral - meaning that the net charge is zero - meaning that the groups are COO- and NH3+
An amino acid will have 3 pka values (except for glycine which has 2). Lets call them pka 1, pka2, pka3. They of course have their own corresponding pH value or [H+]
Pka1 corresponds to the carboxyl group, Pka2 corresponds to the R group, Pka3 corresponds to the amino group. It is possible for the R group to be pka3 and amino group to be pka2, but, you would be told this is the question through words or a graph. However, lets assume what I said earlier corresponds to the protein we are discussing. An increasing pH will encounter pka1, pka2, and pka3 respectively.
A pH below pka1 means that the carboxly group is protonated. This is because many H+ are present in the relatively acidic solution. Because we are also below the other pka values, this means that all of the other groups are also protonated. By the way I am speaking relatively meaning that the concept is basically the same for all amino acids. It is just that the pka(s) differ in their value.
Between pka1 and pka2: the carboxyl group is deprotonated while the R group and amino groups are protonated.
Between pka2 and pka3: the carboxyl group again is deprotonated, the R group is deprotonated and the amino group is protonated.
Above pka 3: Everything is deprotonated including the amino group.
If the carboxyl group is deprotonated it is -1 charge. If the amino group is protonated, it has a +1 charge. The R group of course depends on the group or if it even has an amino group or carboxyl group as aliphatic R groups have no effect of the pka graph.
Determining charges of amino acids depends of the pH the AA is in. For example, "negatively charged and positively charged" AA are called that because they are at physiological pH (7.4pH). So, to assign charges you must look at all the pkas with respect to the pH of the solution and evaluate which ones will be positive, negative or neutral and finally add up the charges
I would like to bring up a question: An AA, with a R group that is amino or carboxyl, at it's pI has a COO- groups and NH3+ group; is the R at it's pI? I believe it is. Please correct me if I am incorrect on something above. Thanks
An amino acid will have 3 pka values (except for glycine which has 2). Lets call them pka 1, pka2, pka3. They of course have their own corresponding pH value or [H+]
Pka1 corresponds to the carboxyl group, Pka2 corresponds to the R group, Pka3 corresponds to the amino group. It is possible for the R group to be pka3 and amino group to be pka2, but, you would be told this is the question through words or a graph. However, lets assume what I said earlier corresponds to the protein we are discussing. An increasing pH will encounter pka1, pka2, and pka3 respectively.
A pH below pka1 means that the carboxly group is protonated. This is because many H+ are present in the relatively acidic solution. Because we are also below the other pka values, this means that all of the other groups are also protonated. By the way I am speaking relatively meaning that the concept is basically the same for all amino acids. It is just that the pka(s) differ in their value.
Between pka1 and pka2: the carboxyl group is deprotonated while the R group and amino groups are protonated.
Between pka2 and pka3: the carboxyl group again is deprotonated, the R group is deprotonated and the amino group is protonated.
Above pka 3: Everything is deprotonated including the amino group.
If the carboxyl group is deprotonated it is -1 charge. If the amino group is protonated, it has a +1 charge. The R group of course depends on the group or if it even has an amino group or carboxyl group as aliphatic R groups have no effect of the pka graph.
Determining charges of amino acids depends of the pH the AA is in. For example, "negatively charged and positively charged" AA are called that because they are at physiological pH (7.4pH). So, to assign charges you must look at all the pkas with respect to the pH of the solution and evaluate which ones will be positive, negative or neutral and finally add up the charges
I would like to bring up a question: An AA, with a R group that is amino or carboxyl, at it's pI has a COO- groups and NH3+ group; is the R at it's pI? I believe it is. Please correct me if I am incorrect on something above. Thanks
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How I break this down is treating the pKa and the pH on similar scales. Frankly, I just compare the pKa to the pH as if it was another pH value.
For example, if you have glycine in physiological pH (7.4), it will be neutrally charged (a zwitterion). Why?
The pKa of the carboxylic acid terminal is between 2-5 and the pKa of the amino terminal is around 9. These are the only 2 protic regions, so they are the only regions with pKa values.
When the pH is greater than the pKa, that region is "acting" as an acid, donating it's protic hydrogen. So in this example the carboxylic acid terminal, with a pKa lower than the pH, would donate it's protic hydrogen and become negatively charged.
Conversely, the amino group with a pKa around 9 is going to become protonated and carry a positive charge because it "acting" as a base since the pH of the solution is lower, i.e. more acidic.
The most accurate way to understand these changes is by using the Henderson-Hasselbach equation, but I've found that that is not always the quickest way. In that case, thinking about if the pKa is lower than the pH (and so that region will be deprotonated) or if the pKa is higher than the pH (and so that region will be protonated) is typically faster.
For example, if you have glycine in physiological pH (7.4), it will be neutrally charged (a zwitterion). Why?
The pKa of the carboxylic acid terminal is between 2-5 and the pKa of the amino terminal is around 9. These are the only 2 protic regions, so they are the only regions with pKa values.
When the pH is greater than the pKa, that region is "acting" as an acid, donating it's protic hydrogen. So in this example the carboxylic acid terminal, with a pKa lower than the pH, would donate it's protic hydrogen and become negatively charged.
Conversely, the amino group with a pKa around 9 is going to become protonated and carry a positive charge because it "acting" as a base since the pH of the solution is lower, i.e. more acidic.
The most accurate way to understand these changes is by using the Henderson-Hasselbach equation, but I've found that that is not always the quickest way. In that case, thinking about if the pKa is lower than the pH (and so that region will be deprotonated) or if the pKa is higher than the pH (and so that region will be protonated) is typically faster.
Thank you for the explanations above.
I understand the concept of pI at the aa level but I am having some trouble calculating it for a protein. Can we work through an example?
Example - what is the pI for the protein Cys-Gly-Glu-Lys-Ala?
I understand the concept of pI at the aa level but I am having some trouble calculating it for a protein. Can we work through an example?
Example - what is the pI for the protein Cys-Gly-Glu-Lys-Ala?
These are the ways to calculate the pI of that polypeptide or another kind of protein:
First "they" will have to provide to pka values for each of the groups capable of acid base reactions. So we have Glu, Lys R groups that will part take in the reaction and the N terminus of Cys and the C terminus of Ala. Overall, we have 4 groups capable of acid base reactions. By comparing all of the pkas you can determine the pH where all of the groups charges (or lack thereof) add up to zero. It might be helpful to draw a titration graph with the pka values are write the charges of the four groups in-between each of the pkas. Eventually you will find a region of the graph where the peptide will have a net charge of zero at a certain pH; this is the pI. --They must give you the pkas, however. Someone could correct me if I'm wrong but I believe the pkas of AA will be slightly different when incorporated into peptides.
The other way is to do this experimentally. This is called isoelectric focusing. A gradient of pH is present on a gel while an electric current is passed through perpendicular to the gradient. When the proteins reach a pH where their pI is zero, the electric current has no force on the peptide. The location's pH is the pI for that particular protein/peptide.
They won't really ask questions directly like this, but, knowing they concepts are key.
First "they" will have to provide to pka values for each of the groups capable of acid base reactions. So we have Glu, Lys R groups that will part take in the reaction and the N terminus of Cys and the C terminus of Ala. Overall, we have 4 groups capable of acid base reactions. By comparing all of the pkas you can determine the pH where all of the groups charges (or lack thereof) add up to zero. It might be helpful to draw a titration graph with the pka values are write the charges of the four groups in-between each of the pkas. Eventually you will find a region of the graph where the peptide will have a net charge of zero at a certain pH; this is the pI. --They must give you the pkas, however. Someone could correct me if I'm wrong but I believe the pkas of AA will be slightly different when incorporated into peptides.
The other way is to do this experimentally. This is called isoelectric focusing. A gradient of pH is present on a gel while an electric current is passed through perpendicular to the gradient. When the proteins reach a pH where their pI is zero, the electric current has no force on the peptide. The location's pH is the pI for that particular protein/peptide.
They won't really ask questions directly like this, but, knowing they concepts are key.
