Amino Acids May Have Positive, Negative, or Zero Net Charge

In aqueous solution, the charged and uncharged forms of the ionizable weak acid groups —COOH and —NH3 + exist in dynamic protonic equilibrium:

While both R—COOH and R—NH3 +are weak acids, R—COOH is a far stronger acid than R—NH3 +. Thus, at physiologic pH (pH 7.4), the α-carboxyl groups of free amino acids exist almost entirely as R—COO− and α-amino groups predominantly as R—NH3 +. The same is true for the side chain carboxyl groups of aspartic acid and glutamic acid as well as the ε-amino group of lysine. Other ionizable groups include the sulfhydryl group of cysteine, the imidazole ring of histidine and the guanidino group of arginine (Figure 1). Figure 2 illustrates the effect that the pH of the aqueous environment has on the charged state of aspartic acid.

Fig1. The protonated R groups of histidine (top) and arginine (bottom) are stabilized by electronic resonance.

Fig2. Protonic equilibria of aspartic acid.

Amino acids in blood and most tissues thus should be represented as in A, in the following figure. Note that these ionized yet net neutral molecules, a consequence of their equal numbers of positively and negatively charged groups, are termed zwitterions.

Structure B cannot exist in aqueous solution because at any pH low enough to protonate the carboxyl group, the amino group would also be protonated. Similarly, at any pH sufficiently high for an uncharged amino group to predominate, a carboxyl group will be present as R—COO−. The uncharged representation B is, however, often used when diagramming reactions that do not involve protonic equilibria.

pKa Values Express the Strengths of Weak Acids & Bases

The strengths of weak acids are expressed as their pKa . For molecules with multiple dissociable protons, the pKa for each acidic group is designated by replacing the subscript “a” with a number. The strength of weak bases is generally expressed as the pKa of their protonated, or conjugate acid, form. This enables the relative strengths of all groups capable of dissociably binding proteins to be compared on a single, continuous scale. The terms “weak acid” and “weak base” thus constitute arbitrary labels since in any protonic equilibrium both acidic and basic forms must participate. The net charge on an amino acid—the algebraic sum of all the positively and negatively charged groups present—depends on the pKa values of its functional groups and the pH of the surrounding medium. In the laboratory, altering the charge on amino acids and their derivatives by varying the pH facilitates the physical separation of amino acids, peptides, and proteins.

At Its Isoelectric pH (pI), an Amino Acid Bears No Net Charge

Zwitterions are one example of an isoelectric species—the form of a molecule that has an equal number of positive and negative charges and thus is electrically neutral. The isoelectric pH, also called the isoelectric point or pI, is the pH mid way between pKa values for the ionizations on either side of the isoelectric species. For an amino acid such as alanine that has only two dissociating groups, there is no ambiguity. The first pKa (R—COOH) is 2.35 and the second pKa (R—NH3 +) is 9.69. The isoelectric pH (pI) of alanine thus is

For polyprotic acids, one must first identify the isoionic species. For example, the pI for aspartic acid is

For lysine, Pi is calculated from:

Similar considerations apply to all polyprotic acids (eg, proteins), regardless of the number of dissociable groups present. In the clinical laboratory, knowledge of the pI guides selection of conditions for electrophoretic separations. Electrophoresis at pH 7.0 will separate two molecules with pI values of 6.0 and 8.0, because the molecule with a pI of 6.0 will have a net negative charge, and that with a pI of 8.0 a net positive charge. Similar considerations underlie chromatographic separations on ionic supports such as diethylaminoethyl (DEAE) cellulose (see Chapter 4).

pKa Values Vary With the Environment

The environment of a dissociable group affects its pKa (Table 1). A nonpolar environment, which possesses less capacity than water for stabilizing charged species, thus raises the pKa of a carboxyl group making it a weaker acid, but lowers the pKa of an amino group, making it a stronger acid. Similarly, the presence of an adjacent oppositely charged group can stabilize, or of a similarly charged group can destabilize, a developing charge. Therefore, the pKa values of the R groups of free amino acids in aqueous solution provide only an approximate guide to their pKa values when present in proteins. The pKa of a dissociable R group will depend on its location within a protein. For example, pKa values that diverge from aqueous solution by as much as 3 pH units are common at the active sites of enzymes, while a buried aspartic acid of thioredoxin, has a pKa above 9—a shift of more than 6 pH units!

Table1. Typical Range of pKValues for lonizable Groups in Proteins

The Solubility of Amino Acids Reflects Their Ionic Character

 The charges conferred by the dissociable functional groups of amino acids ensure that they are readily solvated by—and thus soluble in polar solvents such as water and ethanol, but insoluble in nonpolar solvents such as benzene, hexane, or ether.

Amino acids do not absorb visible light and thus are color less. However, tyrosine, phenylalanine, and tryptophan absorb high-wavelength (250-290 nm) ultraviolet light. Because it absorbs ultraviolet light about 10 times more efficiently than either phenylalanine or tyrosine, tryptophan makes the major contribution to the ability of most proteins to absorb light in the region of 280 nm (Figure 3).

Fig3. Ultraviolet absorption spectra of tryptophan, tyrosine, and phenylalanine.

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