H H O H H O H H O H H O H H O H H O O H H O | | = | | = | | = | | = H2O | | = | | = = | | = ...-N-C-C-N-C-C-N-C-C-N-C-C-... -----> ...-N-C-C-N-C-C-OH + NH2-C-C-N-C-C-... | | | | | | | | R1 R2 R3 R4 R1 R2 R3 R4 Figure 1. The breakdown of protein through the hydrolysis of peptide bondsVarious species of organisms cannot synthesize or are not efficient in generating all of the twenty amino acids needed to construct the proteins and enzymes essential for their survival. To sustain growth and to maintain metabolic functions, these amino acids must be provided from outside sources. This can be accomplished by the intake of proteins. Humans are a good example of living organisms that ingest proteins as part of their nutritional requirements. However, protein molecules are generally quite large, and these large molecules cannot be transported across cell membranes for the same reason that you cannot bring an elephant through the door into your dorm room. Some organisms secrete proteolytic enzymes extracellular to break down the protein to its component monomeric amino acid units by hydrolyzing peptide bonds at the end of the polymer chain. A series of shorter polypeptides of different lengths are also formed if the broken peptide bonds are not at the end of the polymer chain. Thus, depending on the location of the attack, proteases can be further classified into exopeptidases (attack on the terminal group) and endopeptidases (attack on internal linkages).
In the previous experiment, the hydrolysis of a protein was monitored with the release of a dye that was bound to the protein. In this experiment, another more accurate and generally accepted color method is introduced. In this method, an organic compound called ninhydrin is reacted with the amino acids released during the hydrolysis of the protein. The original unreacted ninhydrin is yellowish in color, but the reacted product of ninhydrin has a deep purple-blue color. For example, the procedure given at the end of this section yields an absorbance of 0.27 for 1.X10^-4M of glutamic acid. Since ninhydrin does not react with the undegraded protein, one can measure the amino acid concentration by following the development of the purple color by measuring the absorbance of the solution with a spectrophotometer. Because the color intensity is a measure of the amino acid present, the color should intensify as more protein is degraded to amino acid over time. The upper limit in color intensity is reached when all the ninhydrin originally present in the solution has been consumed. Thus, the amount of ninhydrin originally present in the reaction mixture determines the maximum amino acid concentration that can be detected.
1 2 3 4 26 27 28 29 30 AA--AA--AA--AA--...--AA--AA--AA--AA--AA--AA--AA--AA enzyme activity 100 50