Use of o-phthalaldehyde to detect O-phosphorylethanolamine in bacterial lipopolysaccharide

We have developed a method to measure O-phosphorylethanolamine groups in bacterial lipopolysaccharide using a fluorescent reagent, o-phthalaldehyde. The optimal excitation and emission wavelengths were 335 nm and 450 nm, respectively. The reaction was pH-dependent with an optimum at pH 10.5. The maximum fluorescence intensity occurred two min after mixing lipopolysaccharide with the reagent at pH 10.5. The assay was linear over a range of 1 microgram to 100 micrograms of lipopolysaccharide. When we compared the amount of primary amine (as O-phosphorylethanolamine) in native and p-hydroxyphenylacetic acid-derivatized lipopolysaccharide, we found that 97% of amine groups in native lipopolysaccharide were derivatized by p-hydroxyphenylacetic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.