ABSTRACT
An analytical method capable of identifying >30 chlorophyll-related compounds in plant extracts has been developed. The method employs liquid chromatography coupled to UV-vis, MS, and MS/MS detection. It can be applied without modification to analyze natural chlorophyll degradation products and other metalloporphyrines. It was successfully applied to identify chlorophyll derivatives found in rehydrated spinach powder and conventionally canned and Veri-Green-processed beans. In the Veri-Green-processed beans several degradation products were identified that are zinc-containing analogues to the chlorophyll derivatives found in vegetables after conventional canning. They have been characterized by liquid chromatography and mass spectrometry.
Subject(s)
Chlorophyll/analogs & derivatives , Chlorophyll/analysis , Fruit/chemistry , Vegetables/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Food Handling , Mass Spectrometry/methods , Spectrophotometry/methodsABSTRACT
Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.
Subject(s)
Fructans/biosynthesis , Hexosyltransferases/genetics , Hordeum/genetics , Amino Acid Sequence , Carbohydrate Sequence , Cloning, Molecular , DNA, Complementary/genetics , Enzyme Induction , Gene Expression Regulation, Plant , Glycoside Hydrolases/genetics , Hexosyltransferases/biosynthesis , Hexosyltransferases/isolation & purification , Hordeum/enzymology , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Plant Leaves/enzymology , Plant Leaves/metabolism , Plants, Toxic , Protein Conformation , Protoplasts , Recombinant Proteins/biosynthesis , Sequence Analysis , Sequence Homology, Amino Acid , Nicotiana/geneticsABSTRACT
Sucrose:sucrose 6-fructosyltransferase, an enzyme activity recently identified in fructan-accumulating barley (Hordeum vulgare) leaves, was further characterized. The purified enzyme catalyzed the transfer of a fructosyl group from sucrose to various acceptors. It displayed some [beta]-fructosidase (invertase) activity, indicating that water could act as fructosyl acceptor. Moreover, it transferred the fructosyl residue of unlabeled sucrose to [U-14C]Glc, producing [U-14C]sucrose and unlabeled glucose. Most significantly for fructan synthesis, the enzyme used as acceptors but not as donors a variety of oligofructans containing [beta](2->1)- and [beta](2->6)-linked fructosyl moieties. Thus, it acted as a general sucrose:fructan fructosyltransferase. The products formed by the enzyme from sucrose and various purified, structurally characterized oligofructans were analyzed by liquid chromatography and identified by comparison with structurally characterized standards. The results showed that the enzyme formed exclusively [beta](2->6) fructosyl-fructose linkages, either initiating or elongating a fructan chain of the phlein type. We propose, therefore, to rename the purified enzyme sucrose:fructan 6-fructosyltransferase.
