Titration curve, of amino acids

Discovery of thiazolidine-4-carboxylic acids was made accidentally by Birch and Harris [9] during a study of the effect of formaldehyde on the titration curves of amino acids. Later, Schubert [10] explained the formation of thiazolidine-4-carboxylic acid by condensation of cysteine and formaldehyde followed by intramolecular cyclization. Accordingly, a large number of thiazolidine-4-carboxylic acids can be synthesized by condensation of aldehydes and ketones with cysteine and/or pencillamine as shown in Scheme 3. 

Fig.2b. Amino acids. Sample titration curves of amino acids...

An acid-base conjugate pair can act as a buffer, resisting changes in pH. From a titration curve of an acid the inflexion point indicates the pK value. The buffering capacity of the acid-base pair is the ptC 1 pH unit. In biological fluids the phosphate and carbonate ions act as buffers. Amino acids, proteins, nucleic acids and lipids also have some buffering capacity. In the laboratory other compounds, such as TRIS, are used to buffer solutions at the appropriate pH. 

Figure 4.1 Titration curve of aspartic acid, pKv pK2, and pX3 are pk values of the a-carboxyl, -y-carboxyl and a-amino groups, respectively. The fully protonated form is present at a very low pH (e.g., pH 1) the fully deprotonated form is present at a very high pH (e.g., pH 12). The zwitterionic form (net charge 0) is present at pH = 3, which is also the pi.

Figure 4. Titration curves of glutamic acid, glycine, histidine, and lysine. The three amino acids whose titration curves sharply cut the zero-charge-horizontal-line also have a net charge apart from zero in the vicinity of the pH where they cut the zero line, i.e., the pi. That means that they have buffering capacity and conductance in the neighborhood of the isoelectric point and therefore are useful as carrier ampholytes. Glycine, on the other hand, with its extended horizontal part of the curve is not suitable. (Svensson, 2.)...

The titration curve of penicillamine hydrochloride at 25 °C revealed the presence of three ionizable groups with pKa values of 1.8 (carboxyl group), 7.9 (oc-amino group), and 10.5 (/J-thiol group). Recently, the ionization constants for the acidic functions of (D)-penicillamine were verified by pH titration at 37 °C and 0.15 M ionic strength [2], A 1% solution in water has a pH of 4.5-5.5 [3],... 

Titration Curves Predict the Electric Charge of Amino Acids... 

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... 

In nitrogen heteroaromatics, upfield protonation shifts are found for carbons a to nitrogen, while those in / and y positions are deshielded on protonation [94, 99,100]. This is shown in Fig. 3.5 for quinoline [94]. The protonation shifts for C-/1 and C-y can be rationalized in terms of the cannonical formulae of protonated pyridine [73 d], while the upfield shifts for C-a are probably due to the lower n character of the N — C-a bond. The curves in Fig. 3.5 representing the pH dependence of 13C shifts resemble titration curves. pK values and, in the case of amino acids, the isoelectric points pi can be obtained from the point of inflection of the (5 versus pH plot for each individual carbon [84, 94, 98]. 

Titration curve of /3-lactoglobulin. At very low values of pH (<2) all ionizable groups are protonated. At a pH of about 7.2 (indicated by horizontal bar) 51 groups (mostly the glutamic and aspartic amino acids and some of the histidines) have lost their protons. At pH 12 most of the remaining ionizable groups (mostly lysine and arginine amino acids and some histidines) have lost their protons as well. 

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. 

Figure 3.2 Titration curves for several acids. We are titrating 25 ml of 0.1 M acid with 0.1 M NaOH. Note that H3P04 has three plateau regions, and therefore, phosphoric acid may serve as a buffer at three pH ranges, 1-3, 5.7-7.7, and 11-13 for example, an amino acid, glycine, has two plateau (buffering regions). The numbers at the inflection points of each plateau region indicate the pK values.

The acid-base behavior of amino acids may also be illustrated via titration curves. If one started with aspartic acid hydrochloride, that is, aspartic acid crystallized from solution in hydrochloric acid, one would require 3 mol base to remove completely the protons from 1 mol aspartic acid. The titration curve obtained with structures at each step of the reaction series is shown in Figure 4.1. Note that the isoelectric point is attained after one proton equivalent has been removed from the molecule. At this point, aspartic acid contains one positive and one negative charge it is zwitterionic. 

The titration curves of these amino acids have an extra inflection, as shown for glutamic acid in Fig. 3-3. 

To determine the true titration curve of any substance, you must measure how much acid or base is consumed in titrating the solvent (water) to each pH and then subtract this amount from the total amount of acid or base consumed in reaching that pH when titrating the sample (amino acid + water). The following example with the acid side of the titration of an amino acid illustrates the method for correcting for such acid or base dilution that you should use in correcting your data. 

Within the limits of error of amino acid analyses available at the time, the count of groups obtained by Cannan et al. agreed with expectation, except in so far as the alkaline part of the curve was concerned. The number of groups titrated here is essentially the same as the number of amino groups, rather than the sum of amino and phenolic groups. This result is in accord with the later spectrophotometric titration of phenohc groups essentially all of these groups are inaccessible to titration in the native protein. 

The titration curve of ribonuclease (Tanford and Hauenstein, 1956b) is reversible between its acid end point and the onset of alkaline denaturation. All titratable groups, which are expected to be present on the basis of amino acid analysis, are found to be titrated in the expected parts of the titration curve, with the exception of the abnormal phenolic groups mentioned above. The amino and imidazole groups appear to have normal pK s, and the neutral and alkaline regions in which they occur are compatible with the same values of w as are required to fit the titration curves of the three normal phenolic groups. 

Titration curves of trypsin were obtained under a variety of conditions by Duke et al. (1952). The most noteworthy feature is a specific effect of calcium, which displaces the acid part of the titration curve to lower pH, and decreases the total number of groups which are titrated between pH 6 to 9. It is likely that the groups titrated between pH 6 and 9 in the absence of Ca " are a-amino groups, produced by self-digestion of the enzyme. The effect of Ca" " thus appears to result from a complex with the carboxyl groups of the protein, which stabilizes the anionic form of these groups so as to produce the displacement of the acid part of the titration curve. This complex is more resistant to self-digestion than the enzyme alone. 

Hydrogen ion titration curves of proteins provide a powerful tool to reveal many aspects of the structures of individual proteins. The characteristic ionization constants of the acidic and basic groups in the amino acids and peptides may be profoundly modified when these groups are incorporated in a protein molecule. An increasing number of proteins have been found in which potentially reactive groups are inaccessible for titration in the native molecule, and become available only after denaturation. Such findings can, in the years ahead, be correlated with detailed knowledge of the three dimensional structure of proteins, as obtained by X-ray diffraction and other methods. The present state of the field is reviewed by Tanford, who has done so much to advance it over the last decade. 

The corrected pH-ncutralization curves obtained in this manner are shown by the dotted lines in Fig. 108 the inflexions at the equivalence points are now seen to be sufficiently definite for an accurate estimate of the end-point to be possible. This method has been used for the poten-tiometric titration of amino-acids. ... 

An alternative, simpler, procedure for improving the inflexion in the neutralization of an amino-acid is to add formaldehyde to the solution although this does not affect the acid-titration curve, the one for alkaline titration is changed, as seen in Fig. 107. The effect of the formaldehyde is to increase the strength of the ammonium ion acid which is being titrated, and so the pH inflexion at the equivalence-point becomes much more obvious. This is the basis of the formol titration of amino-acids discovered by Sorensen (1907) approximately 10 per cent of formaldehyde is added to the solution which is then titrated with standard alkali using phenolphthalein as indicator. In the presence of thii concentration of formaldehyde the pH-neutralization curve has a sharp inflexion in the region of pH 9, and so a satisfactory end-point is possible with the aforementioned indicator. 

The pf of amino acids is the pH at one equivalence point along the titration curve, specifically the equivalence point at which all the AA" is converted to AA . The pH at this point is, as usual, the average of the pK value to follow and the p/fo value just passed. Similarly, pH, is the pH at one equivalence point and may be similarly calculated. To determine pJ and pH simply sketch the titration curve and indicate the predominant ionic species present at each key point, Or, prepare a table showing the ionic form of each titratable group at key points. For simplicity, assume that you are starting with the maximally protonated amino acid or peptide. 

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