For instance, if the titration were performed to maintain the pH at 8.4 and the pK of the released amino group was 7.8, it can be shown from the Henderson-Hasselbalch equation that only 0.8 of a proton is released for every peptide bond broken. Acetylation of Amino Groups in Proteins Modification of the functional groups present in the side chains of the amino acid residues in a protein has been used as a device for establishing the importance of these groups in the determination of the biological functions of proteins. There are a variety of reagents available for specific reactions with these functional groups. One of the simplest to use is acetic anhydride, a reagent for the acetylation of free amino groups. In the experiments outlined below, this reagent is used to modify the susceptibility of BSA to tryptic hydrolysis.
Trypsin cleaves proteins at the carboxyl side of the basic amino acid residues arginine and lysine.
Acetylation of lysine residues blocks tryptic hydrolysis at these positions. Tryptic cleavage then occurs only at arginine residues, and allows a direct estimate of the number of arginine residues per mole of protein. This part of the experiment will take from 4 to 6 continuous hours to complete so plan ahead. The heat denaturation of the BSA and trypsin-catalyzed digestion must be carried out in the same vessel because it is difficult to transfer the denatured BSA that adheres to the glass. Overheating may cause formation of lumps that are slow to digest.