Fluorescamine: a rapid and inexpensive method for measuring total amino acids in nectars

Ecology, Dec, 1995 by Erin O'Reilly, Janet Lanza

1) plants with different pollinators produce nectars with different amino acid concentrations (Baker and Baker 1973, 1983, 1986),

2) nectar feeders can detect amino acids in sugar solutions and often prefer such solutions over sugar-only controls (Inouye and Waller 1984, Lanza and Krauss 1984, Potter and Bertin 1988, Rathman et al. 1990, Alm et al. 1990, Lanza et al. 1993),

3) the presence/absence of individual amino acids appears to be inherited independently (Baker and Baker 1976a), and

4) environmental factors do not appear to control the presence/absence of amino acids in nectars (Baker and Baker 1977) but may influence the concentration of individual amino acids (Smith et al. 1990, Lanza et al. 1995).

Studies of amino acids in plant nectars are dependent on methods to identify and quantify the presence of these compounds (see Dafni [1992] and Kearns and Inouye [1993] for good explanations of various methods). All methods have advantages and disadvantages. The most important methods of amino acid analysis that have been applied to nectars are:

1) Ninhydrin staining with histidine standards (Baker and Baker 1973). In this method, nectar samples are spotted on filter paper and stained with ninhydrin; their color is then compared visually to the color intensity of a serially-diluted histidine solution also spotted on filter paper and stained with ninhydrin. This method is rapid, and can be used with small volumes of nectar (1-3 [[micro]liter]; Dafni 1992). The method provides only a rough estimate of amino acid concentration because quantitation is based on a visual comparison to a serially-diluted standard. Quantitation using spectrophotometry is impractical because large volumes of nectar are required. Another shortcoming of this method is that secondary amines, such as proline and hydroxyproline, are not detected because they produce a yellow product whose color is hidden by the blue produced by the other amino acids.

2) Thin layer chromatography (TLC) of dansylated amino acids (Baker and Baker 1976b). Dansylated amino acids are first separated by TLC. The separated amino acids fluoresce under ultraviolet light and their concentrations can be measured precisely with a filter fluorometer equipped with an automatic TLC scanner (Kearns and Inouye 1993). This technique is quite sensitive - nectar volumes as low as 1 [[micro]liter] and concentrations as low as 10 pmol/L can be analyzed (Kearns and Inouye 1993). However, one drawback of this method is that the presence of leucine can obscure the presence of phenylalanine (Kearns and Inouye 1993). In addition, this method is more time-consuming than the ninhydrin test and requires the use of relatively complex and expensive equipment (e.g., filter fluorometer and TLC scanner).

3) Amino acid analyzers (Rust 1977). Nectar components can be analyzed with an automated amino acid analyzer. As with TLC, this method allows measurement of low concentrations of individual amino acids. However, this method requires maintenance of a specialized and expensive piece of equipment and may require relatively large volumes of nectar ([greater than] 10 [[micro]liter]; J. Lanza, personal observation).

4) High performance liquid chromatography (HPLC). Several HPLC techniques have been developed that allow quantification of amino acid concentrations. Smith et al. (1990) applied one of these techniques (Water's Pico[center dot]Tag) to extrafloral nectar. This method is sensitive and low concentrations of individual amino acids from small volumes of nectars can be analyzed (e.g., 0.1 [micro]mol/mL in as little as 3 [[micro]liter] of Impatiens capensis nectar, Lanza et al. 1995). However, this method also requires maintenance of complex and expensive equipment. In addition, the pre-chromatographic derivatization time is long ([approximately equal to]8 h) as is each chromatographic run ([approximately equal to]90 min [Smith et al. 1990]).

Quick surveys of intra- and interspecific variation of nectar-borne amino acids would facilitate many potential projects. An ideal method of quantification would be rapid, inexpensive, and sensitive. All four methods described above fall short in at least one respect. The simplest, ninhydrin, is rapid and inexpensive but less sensitive than is ideal because it involves a visual comparison. The other three methods are sufficiently sensitive but are time-consuming and expensive because they require maintenance of specialized and complex equipment.

We investigated the use of fluorescamine as a rapid, inexpensive, but sensitive method for measurement of total amino acid concentration in nectars. Fluorescamine is a non-fluorescent compound that reacts with primary amines to form a highly fluorescent product (Udenfriend et al. 1972, Weigele et al. 1972, Bohlen et al. 1973, Harris and Bashford 1987). Although fluorescamine is unstable in water, its hydrolysis product is nonfluorescent and does not interfere with quantitation of amino acids. Fluorescamine's reaction with the amines occurs immediately.