Isoelectric Point Of Histidine

[Brian]

Sinceamino acids, as well as peptides and proteins, incorporate both acidic and basic functional groups, the predominant molecular species present in an aqueous solution will depend on the pH of the solution. In order to determine the nature of the molecular and ionic species that are present in aqueous solutions at different pH's, we make use of the Henderson-Hasselbalch Equation, written below. Here, the pKa represents the acidity of a specific conjugate acid function (HA). When the pH of the solution equals pKa, the concentrations of HA and A(-) must be equal (log 1 = 0).

The titration curve for alanine in Figure \(\PageIndex2\) demonstrates this relationship. At a pH lower than 2, both the carboxylate and amine functions are protonated, so the alanine molecule has a net positive charge. At a pH greater than 10, the amine exists as a neutral base and the carboxyl as its conjugate base, so the alanine molecule has a net negative charge. At intermediate pH's the zwitterion concentration increases, and at a characteristic pH, called the isoelectric point (pI), the negatively and positively charged molecular species are present in equal concentration. This behavior is general for simple (difunctional) amino acids. Starting from a fully protonated state, the pKa's of the acidic functions range from 1.8 to 2.4 for -CO2H, and 8.8 to 9.7 for -NH3(+). The isoelectric points range from 5.5 to 6.2. Titration curves show the neutralization of these acids by added base, and the change in pH during the titration.

The distribution of charged species in a sample can be shown experimentally by observing the movement of solute molecules in an electric field, using the technique of electrophoresis (Figure \(\PageIndex2\)). For such experiments an ionic buffer solution is incorporated in a solid matrix layer, composed of paper or a crosslinked gelatin-like substance. A small amount of the amino acid, peptide or protein sample is placed near the center of the matrix strip and an electric potential is applied at the ends of the strip, as shown in the following diagram. The solid structure of the matrix retards the diffusion of the solute molecules, which will remain where they are inserted, unless acted upon by the electrostatic potential.

At pH 6.00 alanine and isoleucine exist on average as neutral zwitterionic molecules, and are not influenced by the electric field. Arginine is a basic amino acid. Both base functions exist as "onium" conjugate acids in the pH 6.00 matrix. The solute molecules of arginine therefore carry an excess positive charge, and they move toward the cathode. The two carboxyl functions in aspartic acid are both ionized at pH 6.00, and the negatively charged solute molecules move toward the anode in the electric field. Structures for all these species are shown to the right of the display.

It should be clear that the result of this experiment is critically dependent on the pH of the matrix buffer. If we were to repeat the electrophoresis of these compounds at a pH of 3.80, the aspartic acid would remain at its point of origin, and the other amino acids would move toward the cathode. Ignoring differences in molecular size and shape, the arginine would move twice as fast as the alanine and isoleucine because its solute molecules on average would carry a double positive charge.

Some amino acids have additional acidic or basic functions in their side chains. These compounds are listed in Table \(\PageIndex1\). A third pKa, representing the acidity or basicity of the extra function, is listed in the fourth column of the table. The pI's of these amino acids (last column) are often very different from those noted above for the simpler members. As expected, such compounds display three inflection points in their titration curves, illustrated by the titrations of arginine and aspartic acid (Figure\ (\PageIndex3\)). For each of these compounds four possible charged species are possible, one of which has no overall charge. Formulas for these species are written to the right of the titration curves, together with the pH at which each is expected to predominate. The very high pH required to remove the last acidic proton from arginine reflects the exceptionally high basicity of the guanidine moiety at the end of the side chain.

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No! Amino acids are acids. They are also bases containing an amino group. The term amphoteric is often used to describe amino acids, meaning that they are capable of acting as both acids and bases.

If the pH is decreased to a low enough value (e.g. pH 1) then the carboxylate salt will be protonated to give the neutral carboxylic acid, and the molecule will have a net charge of +1.

For a typical amino acid, there will be a range of pH values where the positively charged form dominates, another where the neutral form dominates, and finally one where the negatively charged one dominates.

If only there were some formula we could use for figuring out the pH of points A and B on the graph above, where the acid and its conjugate base are present in equal concentration.

There is more to isoelectric point than just the calculation of pI values for individual amino acids! The same concepts also apply to peptides and proteins, each of which will have a pI value that is influenced by the characteristics of its side chains.

The isoelectric point, also referred to as the zwitterion's pH, signifies the pivotal moment when a molecule attains a neutral charge. Picture this: no positive or negative charges prevail, as an equal count of these charged entities coexist harmoniously. When we focus specifically on amino acids, this moment occurs precisely when the molecule's positive and negative charges are perfectly balanced, a delicate equilibrium dictated by the pKa values assigned to the amino and carboxyl groups.

To fully grasp the intricacies of this concept, it is paramount to acknowledge that amino acids form the fundamental building blocks of proteins. These amino acids possess a dual nature, with an amino group (-NH2) on one end and a carboxyl group (-COOH) on the other. These groups exhibit characteristics of weak acids and bases, respectively, and their protonation or deprotonation hinges on the pH of their surroundings. At lower pH levels, the amino group happily embraces protonation (NH3+), while the carboxyl group resists such a transformation (COO-), resulting in an overall positive charge for the molecule. In contrast, at higher pH levels, the amino group stands proud as it sheds its proton (NH2), while the carboxyl group welcomes protonation (COOH), engendering a negative charge for the molecule. However, at the magical isoelectric point, the amino acid stands in its glory as a zwitterion, gracefully adorned with both positive and negative charges.

The isoelectric point serves a significant purpose, serving as a metric to gauge a molecule's acidity or basicity, elements that profoundly influence its solubility, stability, and activity. This value also wields the power to prognosticate the behavior of proteins under diverse conditions, envisioning scenarios involving chromatography or electrophoresis with remarkable accuracy.

The isoelectric point of an amino acid is influenced by its chemical structure, specifically the side chain (R group) that determines its properties. There are 20 common amino acids, each with a unique side chain, and their isoelectric points range from 5.5 to 12.0. The table below lists the isoelectric points of some of the most common amino acids:

Each amino acid has a different isoelectric point, which is influenced by the number and type of charged groups present in the molecule. Amino acids with acidic side chains, such as aspartic acid and glutamic acid, have low isoelectric points, while those with basic side chains, such as arginine and histidine, have high isoelectric points.

At the isoelectric point, the molecule has no net charge, which means it is not attracted or repelled by charged particles in the environment. This may affect its solubility, as charged molecules tend to dissolve more readily in polar solvents. If the pH of the solution is below the isoelectric point of the amino acid, the molecule will have a net positive charge and will be attracted to negatively charged surfaces, making it less soluble. Conversely, if the pH is above the isoelectric point, the molecule will have a net negative charge and be repelled by a negatively charged surface, also decreasing its solubility.

In addition to solubility, the isoelectric point can affect the behavior of molecules during chromatography and electrophoresis. These techniques rely on the separation of molecules based on their charge and size, and the isoelectric point can be used to predict where molecules will move in these systems. For example, during isoelectric focusing, proteins are separated based on their isoelectric point in a pH gradient. As proteins move through the gradient, they will reach a region where the pH is equal to their isoelectric point, causing them to stop moving because they have no net charge and thus can be separated based on the protein's isoelectric point.

Detecting the isoelectric point accurately is crucial in various biological and analytical applications. One widely used technique for this purpose is Capillary Isoelectric Focusing (cIEF) technology. cIEF utilizes the principle of electrophoresis to separate and analyze proteins based on their isoelectric points. In this method, a capillary tube is filled with a pH gradient, typically created using a mixture of carrier ampholytes or immobilized pH gradient (IPG) strips. The protein sample is then introduced into the capillary tube and subjected to an electric field. As the electric current is applied, proteins migrate through the pH gradient until they reach a pH value matching their isoelectric point. At this point, the proteins cease to migrate further due to their neutrality, resulting in their sharp focusing at specific positions along the capillary. The separated protein bands can be visualized using various detection methods such as UV absorbance, fluorescence, or mass spectrometry. cIEF technology offers high resolution, sensitivity, and automation, making it a valuable tool for precise determination and characterization of isoelectric points in complex protein mixtures.

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