We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190 000 theoretical plates are obtained for fluorescently labeled ovalbumin.

The central paradigm of chemistry is that molecular structure determines molecular function. Details of this paradigm can be tested with single-molecule enzymology, where the activity of individual molecules is studied. In all cases reported thus far, there is a large molecule-to-molecule heterogeneity in activity and activation energy. This heterogeneity must arise from differences in structure. Replicate incubations on the same molecule yield consistent results; the structural heterogeneity must be stable over the time period of the experiment, which can extend over several hours. In this paper, we demonstrate that highly purified molecules of bacterial alkaline phosphatase generate identical activity; structurally identical molecules behave identically. In contrast, the glycosylated mammalian enzyme demonstrates a complex isoelectric focusing pattern and has a dramatic molecule-to-molecule variation in activity and activation energy. Glycosylation affects both the kinetics and energetics of this enzymatically catalyzed reaction.

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