Download Amino Acid Composition Analysis of Proteins: Hydrolysis, Separation, and Quantitation and more Study notes Biochemistry in PDF only on Docsity! Today I want to consider methods used to determine the amino acid composition of proteins, i.e., the number of each amino acid in a protein. Determining the a.a. composition involves three basic steps as indicated in the handout. The first step is hydrolysis of the protein to its constituent a.a.. Hydrolysis refers to cleavage of the peptide bonds that is catalyzed by strong acid, strong base or proteins called peptidases. For acid hydrolysis, the protein is dissolved in 6N HCl, sealed in an evacuated glass tube and heated to 100° for 20 hrs- Under these conditions trp is largely degraded. Asn and gln are converted to asp and glu plus NH4+ as a result of the hydrolysis of the amide group- Base hydrolysis is carried out in 2-4 N NaOH at 100° for 4-8 hrs. Under these conditions cys, ser, thr and arg are destroyed. Base hydrolysis can be used to measure the trp content. A mixture of various endo- and exopeptidases can also be used to completely hydrolyze a protein without a change in the a.a.. The conc. of peptidases must be kept low (<1% by weight) since the hydrolysis of a peptidase molecule by other peptidase molecules in solution will also contribute to the mixture of a.a.. Note as expected that asp is eluted first because it is the most acidic a.a. (lowest pI) and therefore the least positively charged at any pH. Arg is eluted last because it is the most basic (highest pI) and therefore the most positively charged at any pH. The third step in a.a. analysis involves quantitating the amount of each a.a. as it comes out of the column. In automated a.a. analysis the effluent is continuously mixed with a solution of ninhydrin which reacts with a.a. and ammonia to form a purple-blue compound as shown in Fig T7.2. The colored solution is passed through a flow cell of a spectrophotometer to record the absorbance which is proportional to the concentration of the a.a.. The amount of each a.a. is determined from the area under each peak of the absorbance vs time elution curve shown in Fig T7.1. More recently a.a. are derivatized with o- phthalaldehyde and 2-mercaptoethanol as shown in the handout and then separated by reversed phase chromatography. Separation is based on differences in solubility of the a.a. derivatives in a nonpolar solvent relative to a polar solvent. The resin has nonpolar groups on its surface and will have nonpolar solvent molecules surrounding it forming a nonpolar stationary phase. The moving phase which carries the a.a. through the resin is more polar.
