Carbohydrate–Lipid Interactions: Affinities of Methylmannose Polysaccharides for Lipids in Aqueous Solution

The interactions between 3- O-methyl-mannose polysaccharides (MMPs), extracted from Mycobacteri- um smegmatis (consisting of a mixture of MMP-10, -11, -12 and -13) or ob- tained by chemical synthesis (MMP-5s, -8s, -11s and -14s), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 258C and pH 8.5 were quantified by electrospray ionization mass spectrom- etry (ESI-MS). Association constants (Ka) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans-parinaric acid) were measured by using an indirect ESI-MS assay, the "proxy protein" method. The Ka values are in the 10 4 -10 5 m � 1 range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the meas- ured affinity of the extracted MMPs for trans-parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly de- veloped competitive binding assay, re- ferred to as the "proxy protein/proxy ligand" ESI-MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affini- ties of approximately (5 � 10 4 ) m � 1 for the mixture of NAs tested. This repre- sents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here repre- sents a novel method for probing car- bohydrate-lipid interactions.