A novel high-performance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3,6-anhydrogalactose.

A novel method has been developed to determine the sugar composition of 3,6-anhydrogalactose-containing polysaccharides, such as carrageenan and agar. The method is based on reductive hydrolysis with a methylmorpholine-borane complex in the presence of acid and subsequent high-performance anion-exchange chromatography analysis of the alditols without any derivatization. The method was validated by 13C NMR analysis of six carrageenans and three agars and by a previously used method based on derivatization to alditol acetates and gas-liquid chromatography analysis. The new method was found to be superior to the gas-liquid chromatography method as the analysis time was less than half. Also it was found to be more accurate and reproducible and no derivatization was required. The analysis of the six different carrageenan samples revealed that homogeneous mu- and nu-carrageenan, theoretically without 3,6-anhydrogalactose residues, cannot be isolated from red seaweeds. Consequently, the question arose if mu- and nu-carrageenans at all are present in seaweeds and if the current hypotheses regarding biosynthesis of carrageenans in the seaweeds are correct. The data demonstrated that carrageenans are highly complex natural polysaccharides, which are more irregular than assumed hitherto. The new analytical technique will permit elucidation of the detailed structure of seaweed polysaccharides and determination of their structure-property relationships.

Proline pipe helix: structure of the tus proline repeat determined by 1H NMR.

The structure of a 22 amino acid peptide, TPPI [Nedved, M. L., Gottlieb, P. A., & Moe, G. R. (1994) Nucleic Acids Res. 22, 5024-5030], that is similar to the proline repeat segment of the replication arrest protein, Tus, has been determined by 1H NMR in 50% trifluroethanol. The structure is a novel left-handed helix having 5.56 residues per turn and a regular hydrogen bonding network that is limited to one side of the helix and contains a channel that runs down the helix axis. The latter feature gives the structure an overall pipe-like appearance; hence, the structure has been designated a proline pipe helix. The Tus proline pipe is also amphiphilic with one side consisting of proline and other nonpolar residues while the other side contains mostly basic and other polar residues. Tus and several other proteins that contain a similar proline repeat sequence are DNA binding proteins. It is shown here that the proline pipe helix of TPPI can be accommodated within the major grove of B-form DNA in a manner that positions nearly all of the basic residues near phosphate groups in the DNA backbone. The proline pipe helical motif may be a structural element of many other proteins including integral membrane receptor proteins.