Genetics and Biochemistry is becoming a big part of Industrialization and in different and major fields and aspects of life. Human beings start to study and dig deeper and deeper into the things and substances around them. Scientists believed that amino acids are the building blocks of protein and protein makes up all living things. A theory came up that if we are able to determine the exact sequence of the amino acids in a polypeptide chain in proteins, a lot of things can be discovered such as prevention and treatment of certain diseases especially those caused by defective amino acids, and the determination of the possible cause of amino acid defects.
The dehydration reaction that forms the peptide bond, the structure that connects amino acids to form a sequence, can be reversed by a reaction called hydrolysis. Peptide bonds of a polypeptide chain can be broken by a strong acid solution at 43 degree Celsius or at 110 degree F. The hydrolysis of the peptide bonds then causes the release of the individual amino acids comprising the chain.
After hydrolysis, samples must undergo two steps to identify and determine the quantity of the amino acids in the sample. The first step is the Ionic Exchange Chromatography and followed by the second step which is Spectrophotometry which is the absorption of light through an apparatus called Spectrophotometer.
In the Ionic Exchange Chromatography, acidic solutions, amino acids have positive charges and become bound to a negatively charged resin in a chromatographic apparatus. The solutions of varying pH and ionic strength cause the amino acids to become negatively charged and released from the resin. Each type of amino acid is then identified by the specific combination of pH and ionic strength at which it is released. Each of the different types of amino acids proceeds to the second step to identify which of the amino acids are in the original polypeptide.
In Spectrophotometry, amino acids are labeled with Ninhydrin using heat. A Spectrophotometer measures the absorbency of each labeled amino acid. The amount of light absorbed by each type of amino acid is proportional to the amount of that amino acid in the original polypeptide chain. The more abundant the amino acid is in the polypeptide chain, the more light it absorbs in the Spectrophotometer.
To determine the amino acid sequence of a polypeptide chain, various methods can be used. In the determination of the sequence at the N-terminal, two methods are commonly used. The Sanger's method; Flourodinitrobenzene labels the free amino group on the N-terminal of the polypeptide chain with a dinitrophenyl group. Acid is then used to hydrolyze away the remaining amino acids in the chain and the labeled N-terminal amino acid is identified using Ion Exchange Chromatography. Another method used is the Edman's method. Using Edman's reagent to label the amino acids, it then removes these amino acids from the rest of the chain. The process is repeated until the entire amino acid sequence is determined.
On the C-terminal of the polypeptide chain, amino acid sequence can be determine by Hydrazine and Carboxypeptidase. Hydrazine binds to the amino acids in the peptide bond but not to the amino acids at the end of the C-terminal; the last amino acid can then be released and identified. Carboxypeptidase, a zinc protease, is an enzyme that cleaves the peptide bonds sequentially starting at the C-terminal.
Larger polypeptides can be cleaved into smaller fragments, and the sequence of these fragments can be determined by using different agents. The Cyanogen Bromide cleaves polypeptides at the carboxyl site of Methionine. Trypsin, a digestive enzyme produced by the pancreas, cleaves polypeptides on the carboxyl side of either Arginine or Lysine. It acts on the amino acids with cationic amino acids and as a serine protease. Another agent is Chymotrypsin which cleaves polypeptides at the carboxyl side of tryptophan, tyrosine, or phenylalanine. It acts as a serine protease as all pancreatic enzymes do.
The quarternary structure of some proteins are composed of subunits made up of more than one kind of polypeptide chain. Denaturing agents disrupt the forces that maintain tertiary and quarternary structure. This separates the different polypeptides so they can be sequenced. The two common denaturing agents used are Urea and Guanadine hydrochloride. After the individual polypeptide chains are separated, they may undergo amino acid analysis.
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