pI and pH relationship in context of ion exchange protein purification - Biology Stack Exchange
Since the original study, similar relationships between the pI of proteins and the buffer pH of successful crystallization experiments have been noted for both. The isoelectric point or pI of an amino acid is the pH at which an amino acid has a net charge of zero. Looking at this standard drawing of an. Most people know that pH is a measure of acidity, but what is acidity? The Brønsted-Lowry theory tells us that an acid is a chemical that donates a proton (H ⁺).
As the pH is increased with the addition of a strong base like sodium hydroxide NaOHthe equilibrium will now shift to the right. As the pH increases, equilibrium will continue to shift to the right, favoring the deprotonated acetate form over the protonated acetic acid. What is the turning point of this equilibrium? Since acetic acid is considered a weak acid strong within the scale of weak acidsthere is a range at which the acetic acid and acetate will form a stable buffer.
Methylamine is amphiprotic, meaning that it can donate or accept a proton thus acting as an acid or a base. In fact, since you are provided with pKa acid values, we have to look at this molecule as the reaction between the protonated amine and its deprotonated conjugate base methylamine. The pKa value given for the amino group on any amino acid specifically refers to the equilibrium between the protonated positive nitrogen and deprotonated neutral nitrogen.
The pKa of the protonated methylamine conjugate acid is like this: As the pH rises towards the pKa value, there will be deprotonation. As the pH continues to rise, more and more molecules will deprotonate till the neutral uncharged form dominates. Finding Charge on Amino Acids in Preparation for Isoelectric Point Calculations While we started by analyzing acetic acid and methylamine independently, the same concept applies when analyzing the amino and carboxyl groups on an amino acid.
The key to understanding isoelectric point is to understand how to find what the charge is at any pH, including when the net charge is zero. With just a hydrogen in place of its variable group, we only have the backbone to examine.
As we analyze the structure of glycine at different pH levels, we see only two values, one each for carboxyl and amino groups, on the amino acid pKa table.
Since pKa relates to an equilibrium constant, you will always have one more structure than the number of pKa values; for example, if there were two pka values, we would expect three structures. When the pH is considerably lower than the pKa we expect both sides to be fully protonated. When we raise the pH a few units above the first pKa, and still well below the second pKa value, the carboxyl group will lose its proton; however, the amino group is still protonated. This is the zwitterion form, with a positive and negative to cancel out.
When you raise the pH to well above the amino value, the nitrogen will lose its proton and thus its charge. We now have negative and zero for a net charge of The zwitterion form can exist anywhere between the the 2 pKa values. So how does this relate to the isoelectric point? Do we randomly pick a value?
As explained in the buffer video abovewhen the pH is exactly at the pKa value, we have an ideal buffer where the molecules exist in equilibrium. Now if we raise the pH to 9.
Amino Acid Charge in Zwitterions and Isoelectric Point MCAT Tutorial -
The isoelectric point is the average of the 2 pKa values that have a neutral molecule as one of its equilibrium species. In other words, find the pKa that takes the amino acid from neutral to -1 9. This sounds like a great deal of work for an amino acid with just 2 side chains. This is especially critical when dealing with acidic or basic amino acids that have a third pKa value for their side chain.
Do we take the average of all three? If just two, which two? Find the pKa which represents the equilibrium between the positive and neutral form. Find the pKa which represents the equilibrium between the negative and neutral form. And average those two.
Since 1 is less than every given pKa, we have too many protons in solution and EVERY potential group will be protonated. This pKa should automatically pop out at you as the pKa between zero and positive 1.
Now raise the pH to 3. Carboxylic Acid Esterification Amino acids undergo most of the chemical reactions characteristic of each function, assuming the pH is adjusted to an appropriate value. Esterification of the carboxylic acid is usually conducted under acidic conditions, as shown in the two equations written below. Under such conditions, amine functions are converted to their ammonium salts and carboxyic acids are not dissociated.
The first equation is a typical Fischer esterification involving methanol. The initial product is a stable ammonium salt. The amino ester formed by neutralization of this salt is unstable, due to acylation of the amine by the ester function.
The second reaction illustrates benzylation of the two carboxylic acid functions of aspartic acid, using p-toluenesulfonic acid as an acid catalyst. Amine Acylation In order to convert the amine function of an amino acid into an amide, the pH of the solution must be raised to 10 or higher so that free amine nucleophiles are present in the reaction system.
Carboxylic acids are all converted to carboxylate anions at such a high pH, and do not interfere with amine acylation reactions. The following two reactions are illustrative. In the first, an acid chloride serves as the acylating reagent. This is a good example of the superior nucleophilicity of nitrogen in acylation reactions, since water and hydroxide anion are also present as competing nucleophiles.
A similar selectivity favoring amines was observed in the Hinsberg test. The second reaction employs an anhydride-like reagent for the acylation. This is a particularly useful procedure in peptide synthesis, thanks to the ease with which the t-butylcarbonyl t-BOC group can be removed at a later stage.
A Quick Guide to pH, pKa and pI - Bitesize Bio
The Ninhydrin Reaction In addition to these common reactions of amines and carboxylic acids, common alpha-amino acids, except proline, undergo a unique reaction with the triketohydrindene hydrate known as ninhydrin. Among the products of this unusual reaction shown on the left below is a purple colored imino derivative, which provides as a useful color test for these amino acids, most of which are colorless.
A common application of the ninhydrin test is the visualization of amino acids in paper chromatography. As shown in the graphic on the right, samples of amino acids or mixtures thereof are applied along a line near the bottom of a rectangular sheet of paper the baseline. The bottom edge of the paper is immersed in an aqueous buffer, and this liquid climbs slowly toward the top edge.
As the solvent front passes the sample spots, the compounds in each sample are carried along at a rate which is characteristic of their functionality, size and interaction with the cellulose matrix of the paper.
Isoelectric point and zwitterions
Some compounds move rapidly up the paper, while others may scarcely move at all. The ratio of the distance a compound moves from the baseline to the distance of the solvent front from the baseline is defined as the retardation or retention factor Rf.
Different amino acids usually have different Rf's under suitable conditions. To animate this diagram Click on It. Oxidative Coupling The mild oxidant iodine reacts selectively with certain amino acid side groups. These include the phenolic ring in tyrosine, and the heterocyclic rings in tryptophan and histidine, which all yield products of electrophilic iodination.
In addition, the sulfur groups in cysteine and methionine are also oxidized by iodine. Quantitative measurement of iodine consumption has been used to determine the number of such residues in peptides. The basic functions in lysine and arginine are onium cations at pH less than 8, and are unreactive in that state. Cysteine is a thiol, and like most thiols it is oxidatively dimerized to a disulfidewhich is sometimes listed as a distinct amino acid under the name cystine.
Disulfide bonds of this kind are found in many peptides and proteins. For example, the two peptide chains that constitute insulin are held together by two disulfide links. Our hair consists of a fibrous protein called keratin, which contains an unusually large proportion of cysteine. In the manipulation called "permanent waving", disulfide bonds are first broken and then created after the hair has been reshaped.
Treatment with dilute aqueous iodine oxidizes the methionine sulfur atom to a sulfoxide. Although this direct approach gave mediocre results when used to prepare simple amines from alkyl halidesit is more effective for making amino acids, thanks to the reduced nucleophilicity of the nitrogen atom in the product.
Nevertheless, more complex procedures that give good yields of pure compounds are often chosen for amino acid synthesis. This procedure, known as the Gabriel synthesis, can be used to advantage in aminating bromomalonic esters, as shown in the upper equation of the following scheme. Since the phthalimide substituted malonic ester has an acidic hydrogen colored orangeactivated by the two ester groups, this intermediate may be converted to an ambident anion and alkylated.