Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Amino Acids
Amino Acids
Electronic Absorption Spectra and Acid-Base Properties of Amino Acids in Solutions - Acid-Base Properties of Amino Acids

In accordance with the acid-base equilibrium principle and depending on the environmental pH, the α-carboxyl and α-amino groups of Amino Acids exist in the following interconvertible forms:

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The state containing both -COOH and -NH₃⁺ groups represents the fully protonated form of The amino acid, which, according to Brønsted, acts as a dibasic acid capable of donating two protons. The dissociation of a dibasic acid occurs in two stages if the pK_a values of the protonogenic groups differ. For Most amino acids, pK_a1(COOH) is around 2, and pK_a2(NH₃⁺) is around 9–10. For Glycine, for example, the dissociation process is expressed by the following equilibrium:

The state with a “+1” charge (-COOH/-NH₃⁺) is observed in strongly acidic environments (pH ≤ 1). In strongly basic environments (pH ≥ 11), the amino acid molecule carries a negative charge of “-1” (-COO^-/NH₂ state). At the pH corresponding to the isoelectric point, the positive charge on the amino group is balanced by the negative charge on the carboxyl group, resulting in a net zero charge for the molecule as a whole. The ranges of existence for Various Forms of specific Amino acids are determined using potentiometric titration data. A typical potentiometric titration curve for an amino acid lacking acidic groups in its R-group is shown in Fig. 1.1.

Fig. 1.1. Potentiometric titration curve of a dibasic acid (amino acid). pK(NH₃), pK(COOH) are the pK_a values for the dissociation of the amino and carboxyl groups, respectively; pI is the isoelectric point. Values in parentheses indicate the net charge (Z) of the amino acid molecule.

Amino acids whose R-groups lack acid-base ionizable groups are referred to as neutral. Among the 20 Proteinogenic Amino Acids, 13 are neutral. The pH value at which an amino acid molecule has a net neutral charge is called the isoelectric point (pH_I or pI). Numerically, pI is equal to the arithmetic mean of pK_a1 and pK_a2.

pI = 1/2(pK_a1 + pK_a2)

At any other point on the potentiometric titration curve (effectively at any pH value other than pH = pI), the amino acid molecule carries a net charge of a certain polarity. The magnitude and sign of this charge are determined by the relative concentrations of the species with “+1” and “-1” charges. For example, at pH = pK_a1, the solution will contain 50% COO^-/NH₃⁺ (zero charge) and 50% COOH/NH₃⁺ (+1 charge). The net charge at this point is +1/2. At pH = pK_a2, it is -1/2. The net charge at any other pH value can be estimated using the Henderson–Hasselbalch equation.

In general, the net charge of an amino acid molecule is determined not only by the α-amino and α-carboxyl groups, but also by the presence of ionizable groups in the R-group. Amino acids with such side chains are listed in Table 1.2.

Table 1.2. Amino acids with ionizable groups in the R-group

The pK_a values for all acid-base ionizable groups of proteinogenic amino acids are given in Table 1.3. Knowing these values makes it possible to calculate the isoelectric points of individual Amino Acids and determine the sign of the charge carried by the molecule at a given environmental pH. This is extremely useful information for optimizing the conditions of certain experiments—for instance, Setting the environmental pH to achieve optimal Separation of amino acid mixtures via Electrophoresis, isoelectric focusing, Ion-exchange Chromatography, etc.

Table 1.3. pK_a values of ionizable groups in proteinogenic amino acids

*- In these amino acids, the R-group ionizes prior to the α-NH₃⁺ group.

The pK_a values for the R-groups in glutamic and aspartic acids (4.25 and 3.86, respectively; see Table 1.3) indicate that these groups are acidic. Consequently, these Two amino acids are classified as acidic. The R-groups of lysine and arginine are basic in character, making them basic amino acids. Positioned between these clearly basic and acidic amino acids are histidine, cysteine, and tyrosine, whose pK_a values are close to 7, providing no strong grounds to classify them definitively as either acidic or basic.

To find the isoelectric point of amino acids with three or more ionizable groups, one calculates the arithmetic mean of the two consecutive pK_a values that bracket the pI point. It is helpful to understand and remember that pI is numerically equal to the pH at which the absolute amounts of positive and negative charges on the molecule are equal—i.e., the molecule has a net zero charge. Once the pI for a specific amino acid is calculated, this relationship allows one to compare it with an experimentally measurable solution pH and estimate which charges (positive or negative) predominate on the molecule at that particular pH. As a reliable rule of thumb, one can safely use the following simple principle: at pH > pI, the molecule acquires a net negative charge, whereas at pH < pI, it acquires a net positive charge. Consequently, when an external electric field is applied (such as during electrophoresis), a molecule in a medium with pH > pI will migrate toward the positively charged electrode (anode), while at pH < pI, it will migrate toward the negatively charged electrode (cathode). This rule applies well to amino acids and relatively small Peptides. However, as the number of amino acid residues in a peptide increases, the error in calculating pI from the pK_a values of individual ionizable groups grows rapidly, rendering the aforementioned method for estimating the net charge sign ineffective or inapplicable.

Below are Examples of pI calculations for several dibasic and tribasic proteinogenic α-amino acids, determined using the pK_a values of their ionizable groups as listed in Table 1.3.

Alanine — a neutral amino acid:

Aspartic acid — an acidic amino acid:

Lysine is a basic amino acid:

To avoid mistakes when selecting the pair of pK_a values required to calculate the pI of a polybasic amino acid or peptide (having more than two dissociable groups), one can simply write out the stepwise ionization sequence for all ionogenic groups within the molecule. When arranging the dissociation steps of an amino acid or peptide, use the pK_a values of the ionogenic groups to order the various forms in ascending sequence of pK_a, starting with the fully protonated form (possessing the highest positive charge). The zwitterionic (isoelectric) form will always lie between the states with charges of (+1) and (-1). All of the above is clearly illustrated by the schemes below for tribasic amino acids (both acidic and basic).