Learning about amino acid structures such as pKa values and protonation states is provided a suitable start to any bio chemist. The protonation states of amino acids are important for understanding enzymatic catalysis, pH-induced conformational changes, and the intermolecular interactions which stabilize the tertiary and quaternary structure of proteins respectively.
Creating a titration curve for a less acidic acid like phosphoric acid or an amino acid (having moderate ph), is basically the undergraduate laboratory experiment. Indeed lots of examples of titration curve exercises can be seen by a simple googling. The amino acid titration gradually explained in a difficult way that the students likely can’t mange which amino acid they are titrating. Instead, students know they have one of four amino acids which they have to perform, that based on the shape of their titration curves. This lab experiment is of extreme level because students must fully exercise their concepts of amino acid chemistry.
This punching experiment is an effective introductory lab for any students in biochemistry field. It familiarizes them with how functional groups give to make a complex titration curve and also how pH can produce effects on protonation state. This experiment needs just a little laboratory equipment including the pH meters and micropipettes respectively. While, the requirements of reagents incorporate concentrated solutions of hydrochloric acid (HCl), sodium hydroxide (NaOH) and the at least four amino acids.
Students initially should take a glance on, lysine, glutamine, glutamic acid, and histidine, because they have almost same molar masses with the similar concentration. This is very much important so that the titration curves can be superimposed to help with the perfect identification of the amino acids.
Nevertheless, each of these amino acid structures has a vary side chain chemistry. Lysine and histidine are mainly basic amino acids, glutamic acid is acidic while the glutamine is a neutral polar amino acid. No doubt variations in the type of amino acid can give different titration curve results.
You have to use each amino acid in this experiment and must have a different side chain chemistry. Glutamine does not compose a titratable R-group and therefore result in a production of distinct titration curve signature from all of the other amino acids who have constituted three titratable groups. Lastly, Histidine composes an extra pKa value has approximately a pH of 6 and is thus can be used to differentiate from the acidic residue Glutamic acid and the basic residue Lysine.
To Determine Titration Curve Of Lysine Procedure:
Amino acid structures labeled A, B, C and D are given and each student is said to prepare 25 mLs of a 20 mM solution of an amino acid means each composition. The amino acids in this laboratory experiment have comparable molar masses and therefore with an of aid the same mass, helps in preparing each amino acid solution irrespective of the identity. For uniformity, all students are guided to utilize the molar mass of lysine.
After measuring the pH meter, a 60 mM HCl solution is utilized to make the pH of each amino acid solution to at least 2 in order to ensure that all the unknowns have the same initial point. Once a pH of 2 is obtained, 1.000 mL aliquots of 50 mM NaOH are poured and the resulting pH is recorded this process is continuous unless the reaction gets stop. However, this process is replicated for each amino acid. All amino acid titrations can be accomplished in at least three-hour laboratory interval.
After all, the students are directed to draw a graph and used to know the identification of each amino acid. By graphing at least three curves the identity of each amino acid structures can be determined. The resultant titration curves which we have analyzed is used for pKa values which assist in the determination of the identity of amino acid-based structures. However, in the final lab, you are again said to draw a graph, and go for the result.
As we seen, each amino acid has a distinct curve graph. The shapes of these curves come to know the students to properly determine the amino acids. Furthermore, this experiment also gives a solid foundation of nature of amino acids and how their protonation states vary at different pH states. Amino acid titration curves play a crucial role to understanding not only how pH puts effects on an amino acid chemistry but are integral to understand protein structure as well.
Each amino acid curve has distinct nature that makes them easily interpretable. Histidine exposes a distinct plateau at pH 6, because of the imidazole chain that is attached to side ways. Via, using the result we can say that Glutamic acid contains two acidic groups. The curves between lysine and glutamine are a bit harder to make a difference. The glutamine has an extra inflection point near pH 11, when the amino group becomes totally deprotonated, whereas a couple of amino groups of lysine mix together for a gradual rise for two identical of the base. Despite the complexities in identification, students are able to appropriately identify the each amino acids that based on the superimposition of the graph- curves.
This experiment successfully shows the capability to analyze a titration curve in order to distinguish each unknown amino acids. Titration curves are really vital for any students to make their concepts clear. This experiment wants an abstract concept that clearly mentioned in most of the General Chemistry and Biochemistry acknowledges textbooks. By matching the titration curves with the four different amino acids students are needed to show the side-chain of amino acid chemistry. Finally, and the most prominent thing that this experiment provides students practical skills like, making solutions and how to use a pH meters etc.