University:
Boston University
Course:
CAS BI 108 | Biology 2
Academic year:

2022
Views:

25

Pages:

9
Author:

Annabel Combs

Reagents for Protein Sequence Determina4on 1. Ninhydrin: for quantitative reaction with amino groups of any amino acid 2. Edman Degradation: for reaction with amino terminus of proteins/ peptides and reduction of peptide by one residue 3. N-terminal analysis: for reaction with amino terminus of proteins/ peptides, but complete hydrolysis of peptide required a. FDNB b. Dansyl chloride c. Fluorescamine d. o-phthalaldehyde Ninhydrin The ninhydrin reaction quantitatively reacts with amino acids in a complex reaction that produces ammonia from the amine, carbon dioxide from the carboxylate, and the Ca becomes an aldehyde of the R-group. The key is the reaction of the intermediate hydrindantin with another mole of ninhydrin and the ammonia to produce the large molecule, called Ruhemann’s purple, which is a deep purple and easily quantified by absorbance at 570 nm. Mechanism: Edman Degrada4on OR Biochemical Reac4ons for N-‐terminal analysis Sanger Reagent (ﬂuorodinitrobenzene-‐FDNB; aka dinitroﬂuorbenzene-‐DNFB) Dansyl chloride The dansyl choride reagent reacts with amino terminal amino acid. Subsequence acid hydrolysis yields all the amino acids plus the N-terminal one modified by the dansyl group. This modified amino acid is highly fluorescent and allows detection from very small amounts of protein Dansyl chloride Another depic4on: Fluorescamine & o-‐Phthalaldehyde Fluorescamine [I] reacts readily with primary amino groups to form highly ﬂuorescent compounds [II]. Even though ﬂuorescamine itself is nonﬂuorescent. The ﬂuorescent products have an excita4on maximum at 390 nm and an emission maximum at 475 nm. These proper4es make ﬂuorescamine ideal for detec4ng amino groups, especially in proteins, pep4des, and amino acids. It is 10 to 100 4mes more sensi4ve in detec4ng primary amino groups than the ninhydrin reac4on. Because ﬂuorescamine has low solubility and stability in water, o-‐phthalaldehyde [III] (OPA), which reacts similarly with primary amino groups and gives highly ﬂuorescent products [IV]. OPA is some4mes subs4tuted for ﬂuorescamine: These reactions need to include either a thiol (β-mercaptoethanol depicted) or cyanide anion. Example of primary-‐ structure determina4on