Alanine titration curve: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: a.
Buffer pairs: The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3+/–NH2 pair can buffer in the region around pK2.
b.
When pH = pK: When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution.
c.
Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) as shown in Figure 1.11. The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1).
Separation of plasma proteins by charge typically is done at a pH above the pI of the major proteins. Therefore, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobility pattern are suggestive of certain diseases.
6. Net charge at neutral pH: At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group (−NH3+), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes).
D. Other applications of the Henderson-Hasselbalch equation
The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding salt form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO3−], and the carbon dioxide concentration [CO2] influence pH (Fig. 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Fig. 1.12B). Acidic drugs (HA) release a H+, causing a charged anion (A−) to form.
Weak bases (BH+) can also release a H+. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B).