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bis102-10-3-lec5

bis102-10-3-lec5 - amino acid sequence...

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10/3/2011 amino acid sequence determination: -isolate and determine its amino acid composition Amino terminal amino acid determination - (label the amino terminal) reagents commonly used: both react with free NH2 groups 1. Sanger reagent - 2, 4- fluorodinitrobenzene (FDNB). After reaction, hydrolyze the peptide and will obtain the DNP alpha-amino acid as well as DNP-lysine where the DNP is on the epsilon amino group. -Has micromolar sensitivity 10^-6. 2. Dansyl chloride - to react with free amino group and make Dansyl amino acid which is fluorescent. This reagent will also react with the alpha amino group as well as the epsilon amino group of lysine. Nanomolar sensitivity - 10^-9 Carboxyl terminal amino acid determination- usually conducted using an enzyme that can specifically remove the COOH terminal amino acid. 1. Carboxylpeptidase A- exopeptidase that can remove all the amino acids except lysine, arginine or proline and will not remove any amino acid acid if proline is the penultimate (next to the last or second form the right)
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