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Calculating Net Protein ChargePowerPoint Presentation

Calculating Net Protein Charge

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Presentation Transcript

The Problem

- A protein’s net charge depends on the number of charged amino acids it contains and the pH of its environment.
- Theisoelectric point is the pH at which the net charge is zero.

- Each amino acid has a different dissociation constant, KD.
- The amino acids that can be ionized at physiological pHs are: aspartate (D), glutamate (E), lysine (K), arginine (R), and histidine (H).
- Also, the N and C termini can be ionized.

- The important point is: most of these amino acids are not fully ionized or fully unionized at physiological pHs.
- It is your job to calculate the proportion of each amino acid that is ionized at a given pH. Once this is done, calculating the net charge is straightforward.
- Multiply the proportion by the number of that amino acid, taking into account the differences between acidic (-COOH) and basic (-NH2) types.

Amino Acids Contributing to Charge

- Charged amino acids come in two forms:
- Acidic amino acids (and the C-terminal) are in the –COOH form (uncharged) at low pH and in the –COO- form (-1 charge) at high pHs.
- These include aspartic acid (D) and glutamic acid (E)

- Basic amino acids (and the N-terminal) are in the –NH3+ form (+1 charge) at low pH and in the –NH2 form (uncharged) at high pH.
- These include lysine (K), arginine (R) and histidine (H).

- All other amino acids do not affect charge under physiological conditions.

Dissociation Constant

- The association and dissociation of the H+ with the amino acid is governed by its dissociation constant, KD.
- Dissociation constant is the ratio of the concentration of the dissociated forms to the concentration of the combined form.
- Each amino acid has a separate dissociation constant.
- The equation below uses [R-] to indicate the concentration of the amino acid when the H+ is dissociated from it.

- The pK value is analogous to pH.
- pK = -log10KD
- KD = 10-pK

Calculating the Proportion with H Associated

We rearrange to get:

- Starting with the dissociation constant equation, we need to derive an equation for the proportion of amino acid molecules that have H associated at a given pH.

Substituting this into the

proportion equation:

Some algebraic manipulation:

We want:

Start with the definition of

dissociation constant:

Our final result:

Acidic and Basic Amino Acids

- The proportion equation calculates the proportion of a given amino acid that has H associated with it at a given pH.
- Recalling that [H+] = 10-pH and pH = -log10[H+]

- For the basic amino acids (H, K, and R) and the N-terminus, the form with H associated is –NH3+, which has a +1 charge.
- Thus, the contribution to net charge for each of these amino acids is just the number of each amino acid multiplied by the proportion that has H associated.

- For the acidic amino acids (D and E) and the C-terminus, the form with H associated is –COOH, which is uncharged. Thus, the proportion that is in the charged form ( -COO-) is 1 minus the proportion with H associated.
- Also, remember that the charged form has a negative charge, The net contribution from acidic amino acids needs to be subtracted from the contribution of the basic amino acids.

Summary

- Calculate the proportion of each ionizable amino acid (plus the N- and C-termini) that have H associated at the given pH.
- For the acidic amino acids, calculate the percentage that are charged by taking one minus the proportion with H associated.
- Multiply the proportion charged by the number of each amino acid present in the protein.
- Subtract the negative charge total from the positive charge total to get the net charge.

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