Purification and properties of a periplasmic D-xylose-binding protein from Escherichia coli K-12.

Escherichia coli contains a cold osmotic shock-releasable binding protein for D-XylOSe which has been purified and characterized. The D-xylose-binding protein has an apparent molecular weight of 37,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis. D-Xylose-binding protein has an apparent absolute specificity for D-XYlose and binds one molecule of D-XylOSe per molecule of protein with a Kd of 0.6 PM. The protein exhibits native tryptophan fluorescence (hex 284 nm, he, 345 nm) and a fluorescence enhancement (32%) associated with D-xylose binding which saturates with a Kd of 0.5 PM. DXylose-binding protein does not bind to a Cibacron blue 3FGA affinity column but does interact with a tetraiodofluorescein-agarose affinity column. D-XYlose-binding protein interaction with tetraiodofluorescein, if present, does not produce a red shift in the absorption spectrum of the dye in contrast to the Larabinoseand D-galactose-binding proteins. Tryptophan oxidation with N-bromosuccinimide results in a concomitant drop in xylose binding. D-Xylose-binding protein contains no cysteine and possesses a PI of 7.4.