Sine A. Johannesen, Ole Hindsgaul
The objective of this project is to develop reliable methods for the addition of a single TAG onto both starch-related and beta-glucan polysaccharides. The method should be quantitative, rapid and allow the installation of a variety of TAGs, depending on the intended use of the polysaccharide. Fluorescent TAGs are immediately useful since they permit spectrophotometric detection and quantitation of the polysaccharide.
We have optimized the protocol summarized in Fig. 1 that permits efficient labelling of both small (maltooligosaccharides) and large polysaccharides (Gn indicates multiple glucose units). The dye used is called Py1 (in blue), and is fluorgenic, meaning that the aminated polysaccharide can be quantitated by fluorescence WITHOUT removing excess Py1. The entire process is done in half a day.
Fig. 1 Reaction scheme
A standard curve has been prepared (Fig. 2), showing that the fluorescence is indeed linear with polysaccharide concentration using maltohexose as a standard. This means that we can visually estimate the number of molecules of polysaccharides that are present in a sample, and quantitate this by a fluoresecent measurement.
Fig. 2 Standard curve (ex 485 nm, em 635 nm).
The Py1 dye was selected not only for the fluorogenic properties just described, but also because it is amphiphilic and imparts a permanent positive charge on the labelled polysaccharide. This suggested to us that this TAG would offer advantages in the analysis of polysaccharides by MALDI-TOF mass-spectrometry. This turned out to be the case, as a commercial maltooligosaccharide fraction (Sigma) was labelled and yielded an extraordinary mass spectrum (Fig. 3), where individual glucose units can be discerned up top a DP of at least 60 (near 10,000 molecular weight).
Fig. 3 MALDI-TOF of Py-1 labelled maltooligosaccharides