Alanine titration

If a sample contains groups that can take up or lose a proton, (N//, COO//), then one must expect the pH and the concentration to affect the chemical shift when the experiment is carried out in an acidic or alkaline medium to facilitate dissolution. The pH may affect the chemical shift of more distant, nonpolar groups, as shown by the amino acid alanine (38) in neutral (betaine form 38a) or alkaline solution (anion 38b). The dependence of shift on pH follows the path of titration curves it is possible to read off the pK value of the equilibrium from the point of inflection... 

Each leg of the titration curve is calculated separately. The first leg, from pH 1 to 6, corresponds to the dissociation of protonated alanine, H2A+. The second leg, from pH 6 to 11, corresponds to the dissociation of zwitterionic alanine, HA. It s as if we started with H2A+ at low pH and then titrated with NaOH. When 0.5 equivalent of NaOH is added, the deprotonation of H2A+ is 50% done when 1.0 equivalent of NaOH is added, the deprotonation of H2A+ is complete and HA predominates when 1.5 equivalent of NaOH is added, the deprotonation of H A is 50% done and when 2.0 equivalents of NaOH is added, the deprotonation of HA is complete. 

Figure 26.1 A titration curve for alanine, plotted using the Henderson-Hasselbalch equation. Each of the two legs is plotted separately. At pH < 1, alanine is entirely protonated at pH = 2.34, alanine is a 50 50 mix of protonated and neutral forms at pH 6.01, alanine is entirely neutral at pH = 9.69, alanine is a 50 50 mix of neutral and deprotonated forms at pH > 11.5, alanine is entirely deprotonated.

Thymine, electrostatic potential map of, 1104 structure of, 1101 Thyroxine, biosynthesis of, 551 structure of. 1020 TIme-of-flight (TOP) mass spectrometry, 417-418 Titration curve, alanine, 1023 TMS, see Tetramethylsilane see Trimethylsilyl ether Tollens reagent, 701 Tollens test, 992... 

Estimate the titration curve for 0.200 L of 0.120 M diprotic alanine with 3.00 M NaOH. 

The first end-point in such a titration is due to the carboxyl group and the pKa value for this is called pKal while the second pK value is for the amino group and is called pKa2. In practice each acid and its salt will act as a buffer over a pH range of approximately one unit on either side of its pKa value. For the amino acid alanine, where pKal is 2.4 and pKa2 is 9.6, the most effective buffering action occurs over the pH ranges 2.4 1.0 and 9.6 1.0. 

B. Although titration curves for proteins are complex because of their multiple acidic and basic groups, their behavior can be illustrated by titration of a simple amino acid such as alanine (Figure 2-1). 

Figure 2-1. Titration of a solution of alanine with a strong base. One equivalent of base is the amount needed to titrate the protons from one group on all the alanine molecules present in the solution. Below the titration curve is a calculation of the pi for alanine derived as the mean of its two pK values.

Dissociation of the carboxyl group The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. Consider alanine, for example, which contains both an a-carboxyl and an a-amino group. At a low (acidic) pH, both of these groups... 

Application of the Henderson-Hasselbalch equation The dissociation constant of the carboxyl group of an amino acid is called K1f rather than Ka, because the molecule contains a second titrat-able group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid. 

Titration curve of alanine By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possi ble 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 ... 

Titration shows the presence of a terminal phosphomonoester residue, and the average chain length is about 18. The d-alanine residues have the expected reactivity toward ammonia, and, from a study of the reaction with hydroxylamine, it is concluded that all of these residues are attached to available 2-hydroxyl groups of glycerol, instead of to the 2-acetamido-2-deoxy-D-galactosyl residues. 

A simple amino acid with a nonionizable R group gives a complex titration curve with two inflection points. For an example, see the titration of alanine, shown in figure 3.3. At very low pH, alanine carries a single positive charge on the a-amino group. The first inflection point occurs at a pH of 2.3. This is the pK for titration of the carboxyl group,... 

Titration curve of alanine. The predominant ionic species at each cardinal point in the titration is indicated. 

As a final example of an acid-base titration, let s consider the gradual addition of NaOH to the protonated form of the amino acid alanine (H2A+), a substance that acts as a diprotic acid. Amino acids (which are discussed in more detail in Chapter 24) are both acidic and basic and can be protonated by strong acids such as HC1, yielding salts such as H2A+C1 . The protonated form of the amino acid has two... 

Lippmaa and coworkers used 13C NMR titration to measure secondary deuterium IEs on acidities of carboxylic acids.24 The results are listed in Table 2. It is remarkable that the IEs show only a small attenuation with distance, so that the IE from three y-deuteriums in alanine is greater than that from two (3-deuteriums in glycine. As a consequence the IEs can be detected and measured from deuteriums as far as seven bonds away from the carboxyl, as in caproic-6,6,6-fl 3 acid. Moreover, in benzoic acids the IE is practically independent of the site of deuteration. 

Thiourea functions were used to attach chiral saccharide units to the molecule of calix[4]arene [72]. The complexation properties of these molecules toward chiral anions have not yet been examined. However, in the preliminary complexation studies (XH NMR titrations in DMSO-d6) the affinity toward acetate and AT-Ac-L-alaninate was observed. 

The lower rim C-linked AT-tosyl peptidocalix[4]arenes [78] of type 60 bearing l- or D-amino acid residues were prepared and studied for their anion recognition ability. The H NMR titrations proved that in all cases only the 1 1 complexes are formed in CDC13 with high complexation constants (e.g. 60,d,l-Ala, n=4, R=Pr K60 (HSO )=3,850 M"1, K60 (AT-tosyl-L-alaninate)=6,900 M-1). 

The most notable difference between the acid and basic forms of the PBP is the conformation of the C-terminus. In the basic form the C-terminus is extended, on the surface of the protein (Sandler et al., 2000), while in the acid form the C-terminus forms a seventh a-helix, which occupies the pheromonebinding cavity. The loop with two vicinal histidine residues (69 and 70) has also moved significantly between the two forms, suggesting that titration of one of these residues has a profound effect on the conformation of the PBP (Horst et al., 2001). Consistent with this, mutation of histidines 69 and 70 to alanine, abolished a conformational change detectable upon ligand binding (Mohl et al., 2002). 

Mixed N- and O-Donor Ligands. Spectrophotometric titration methods have been used to establish the existence and stoicheiometry of V111 complexes with alanine, a-aminobutyric acid, lysine, and glycylglycine pH measurements showed that the amino-acid ligand is co-ordinated in the anion form.385... 

You will obtain a titration curve of an amino acid with a neutral side chain such as glycine, alanine, phenylalanine, leucine, or valine. If pH meters are available, you read the pH directly from the instrument after each addition of the base. If a pH meter is not available, you can obtain the pH with the aid of indicator papers. From the titration curve obtained, you can determine the pK values and the isoelectric point. 

For Questions 4.14-4.17, use the following information. You have isolated an unknown peptide whose titration curve is presented in Figure 4.16. The peptide absorbed ultraviolet light at 280 nm. Treatment with trypsin released free alanine and arginine. Treatment with chymotrypsin resulted in quantitative production of a neutral tripeptide and an alkaline dipeptide. 

The binding affinities of SmeHyuA C76A and C181A active site mutants (alanine mutants) were evaluated for further insight into the role of each residue. Binding of the above substrates to both mutants was also measured by isothermal titration calorimetry which showed that the presence of a proton at the 5-position of the substrate and not in the lateral chain is the critical factor for proper binding. 

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