Free proline content in the leaves is determined following the method off (Bates et al., 1973). The protocol is based on the formation of red colored formazone by proline with ninhydrin in acidic medium, which is soluble in organic solvents like toluene.
Proline (abbreviated as Pro or P) is a α-amino acid, one of the twenty DNA-encoded amino acids found in proteins. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary. The more common L form has S stereochemistry.
Proline estimation is based on the formation of brick red colored praline-ninhydrin complex in acidic medium. This complex is soluble in toluene and thus can be separated from aqueous phase. This ensures that there is no interference with other amino acids, which also form blue colored complex with ninhydrin. The toluene soluble brick-red colored complex absorbs at 520 nm.
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