In the Laboratory
NMR Titration Used To Observe Specific Proton Dissociation in Polyprotic Tripeptides An Undergraduate Biochemistry Lab J. L. Yarger,* R. A. Nieman, and A. L. Bieber Arizona State University, Tempe, AZ 85287 Biochemists and other life scientists are concerned with the dissociation of water and equilibrium processes of protic ionizable groups because the proper functioning of any biological system is critically dependent upon pH (1). The primary effect of hydrogen ion concentration, [H+ ], is to change the net charge of biological molecules by the association–dissociation of protons. The side chains of many naturally occurring amino acids (AA) have dissociable protons. As shown in Figure 1, even simple peptides have multiple acid–base equilibria. Standard acid–base titration, one of the classical techniques, cannot easily resolve these overlapping proton dissociations in most peptides and proteins, and it does not have the sensitivity to allow small quantities to be analyzed. However, nuclear magnetic resonance (NMR) can probe individual protons in small-quantity peptides and small proteins, making it a valuable tool for the study of specific proton association–dissociation equilibrium in complex polyprotic systems. NMR can provide a wealth of information on the dynamic processes of proteins in their natural aqueous environment and is the only technique that can determine polypeptide or protein structure in solution. The procedures and strategies that are common today for studying structure and dynamics of biological systems have been developed in the course of the last twenty years (2). Despite the establishment of NMR in biochemical research, this technique has not been incorporated into many biochemistry laboratory courses. The paucity of advanced biochemistry laboratory experiments motivated us to create this experiment, which illustrates basic 1- and 2-D NMR techniques used to study specific proton dissociation in peptides. Tripeptides were chosen as model compounds in this laboratory exercise because of their solubility and resistance to hydrolysis over a large pH range, low cost of samples, and NMR simplicity. Yet these compounds still demonstrate the basic polyprotic behavior of complex proteins. The proton dissociation of the hydroxyl group of tyrosine is easily monitored from the chemical shifts of the adjacent aromatic proton resonances, which are at lower frequency than the majority of the proton resonances in peptides. Although we have chosen to study peptides containing tyrosine, aromatic resonances can be similarly used to observe protonation of peptides containing histidine. In its most basic form, this experiment probes the effect of neighboring amino acids on the ionization of the tyrosine hydroxyl group. The tyrosyl resonances of the three peptides are readily identified at each pH value, and measurement of the pKs from a plot of chemical shifts as a function of pH is straightforward. In addition, the same NMR spectra can be used to determine *Corresponding author. Current address: Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720.
Figure 1. A palindromic tyrosyl-centered tripeptide (AA-tyr-AA) with the three different amino acids used (R) is shown above. Typical pKa*s are given for the ionizable groups of interest.
the pKs of the terminus NH 3+ and COOH groups, as well as any other dissociation on the amino acid side chains (e.g., the amino pK of lysine). Thus, the difficulty of the exercise can be adjusted without increasing the time required in the laboratory. Typical dissociation constants (pK*) for the various ionizable groups of the individual amino acids found in the tripeptides used here are given in Figure 1. pK a values are dependent on temperature, ionic strength, and the microenvironment of the ionizable group. From the experimental pH titration chemical shifts, apparent pKa values (pKapp) can be determined for a given set of conditions by using the Henderson–Hasselbalch equation:
pK a = pH + log
HA A–
The one-half titration technique, [HA] = [A{] at halfheight of titration curve, is valid for acids with small pKas (
