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

Since 1973, when Herbert and Irene Baker first documented the widespread occurrence of amino acids in plant nectars, a wide variety of studies have shown that:

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.

In order to test if fluorescamine is a good tool for measuring total amino acid content in plant nectars, we experimentally asked four questions: (1) is fluorescamine sufficiently sensitive to measure amino acid content in plant nectars? (2) can fluorescamine be used with the small volumes of nectar that most plants produce? (3) do the high concentrations of sugars in nectars interfere with the fluorescence of amino acid-fluorescamine products? and (4) how much error is introduced into total amino acid concentration measurements as a consequence of the fact that different amino acids fluoresce at different intensities?

Fluorescamine Procedure

1) Collect nectar. It may be stored at -70 [degrees] C until analysis.

2) Measure the volume of nectar to be analyzed.

3) Choose an amino acid and make a serial dilution to be used in generating a standard curve. We recommend histidine, threonine, or isoleucine as standards because they fluoresce at moderate intensities. We recommend concentrations of 0.3-30 mmol/L.

4) Take a volume of each serial dilution equal to that of the nectar sample to be analyzed.

5) Dilute all samples with 750 [[micro]liter] of a borate buffer at pH 8.5. (Our buffer was 3.0 g boric acid and 4.8 g sodium tetraborate in 200 mL of distilled water. This buffer can be stored at 4 [degrees] for several months. Other borate buffers can apparently be used [Udenfriend et al. 1972, Weigele et al. 1972, Bohlen et al. 1973, Harris and Bashford 1987].)

6) While vortexing the sample/buffer tube, add 50 [[micro]liter] of a fluorescamine solution (7.5 mg fluorescamine [Sigma Chemical Company, St. Louis Missouri, Catalog Number F-9015] in 25 mL of acetone). Note that fluorescamine is a light sensitive solution and should be kept in a dark bottle. Vortexing is essential to prevent fluorescamine hydrolysis in water.

7) Wait 2 min and measure fluorescence at 390 nm excitation and 465 nm emission. We developed our procedures using a Perkin Elmer LS5 spectrofluorometer. Subsequently, we have successfully used a Hoefer Scientific Instruments DNA Fluorometer Model TKO 100. Although this model excites the solution at wavelengths of 365 nm and reads at wavelengths of 460 nm, its wavelengths were sufficiently adjustable that we could construct standard curves and measure unknowns.
TABLE 1. Amino acid (AA) and sugar composition of solutions used in
testing fluorescamine.


                                 Solution
                                   P. meni-        Lantana
Solution          Four amino      spermifolia       camara
component            acid           nectar          nectar
(AA/sugar)         mixture         mimic(*)        mimic(**)


                           Concentration (g/100mL)


Alanine                             0.064            0.0064
Arginine                            0.257            0.0032
Aspartate                           0.080            0.0056
Asparagine                          0.077
Cysteine            0.118           0.005
Glutamate                           0.356            0.0048
Glutamine           0.120           3.405            0.0136
Glycine             0.056           0.027            0.0178
Histidine                           0.629
Isoleucine                          0.440
Leucine                             0.512
Lysine              0.120           0.044
Methionine                          0.164
Phenylalanine                       0.898
Proline                             2.293            0.0256
Serine                              0.089            0.0144
Threonine                           0.054            0.0080
Tryptophan                          0.798
Tyrosine                            1.432            0.0040
Valine                              0.240            0.0016
Sucrose                                             18.675
Fructose                                             5.700
Glucose                                              5.580


* Lanza 1991.


** I. Baker, personal communication.


8) Estimate the concentration of the sample by comparing its fluorescence to that of the standard curve. Factor in any dilutions of the nectar that were made.

Methods and Results

In testing the usefulness of fluorescamine for nectar analysis, we used one mixture of four amino acids and two mimics of natural nectars (Table 1). The mimics were based on HPLC analysis of the extrafloral nectar of Passiflora menispermifolia (Lanza 1991) and paper chromatographic analysis of Lantana camara floral nectar (I. Baker, personal communication).

We first tested whether fluorescence was sufficiently sensitive to measure the concentrations of amino acids normally occurring in plant nectar. In this test, we serially diluted a mixture of four amino acids (Table 1) and measured the fluorescence of a 200 [[micro]liter] sample of each dilution [ILLUSTRATION FOR FIGURE 1A OMITTED]. At low concentrations, the fluorescence measurements increased linearly with amino acid concentration; at concentrations [less than]0.2 [micro]mol/mL, however, fluorescence did not increase proportionally with concentration. When analyzing nectars with high concentrations of amino acids, this "quenching" can be avoided simply by using smaller volumes or dilutions of test nectars. Thus, it is important to equalize the volume of (1) the nectar mimic used in preparing the standard curve, and (2) the unknown nectar.

We then used a mimic of P. menispermifolia extrafloral nectar to determine whether the fluorescamine method is sufficiently sensitive to use with the small volume of nectar produced by most plants. We serially diluted the P. menispermifolia mimic and measured the fluorescence produced by 5 [[micro]liter] samples. Using only 5 [[micro]liter] of the nectar mimic, the fluorescamine method accurately measured total concentrations of amino acids as low as 0.4 [micro]mol/mL [ILLUSTRATION FOR FIGURE 1B OMITTED]. This concentration is near the low end of natural nectar concentrations (Baker and Baker 1983); greater volumes of nectar would allow measurement of even lower concentrations.

Next, we tested whether the high concentration of sugars normally present in nectars interfered with measurement of the amino acid concentrations. We made three mimics of L. camara floral nectar, one containing only amino acids, one containing only sugars, and one containing both amino acids and sugars (Table 1). In two replicates, the fluorescence of the sugar-only mimic almost matched the background level of blanks containing only buffer and fluorescamine [ILLUSTRATION FOR FIGURE 2A OMITTED]. In two replicates, fluorescence of the sugar-amino acid mimic was equivalent to that of the amino acid-only mimic [ILLUSTRATION FOR FIGURE 2A OMITTED].

Finally, we estimated the magnitude of the error introduced by differential fluorescence of different amino acids. Some amino acids fluoresce more intensely than others. We measured the fluorescence intensity of all 20 protein amino acids; in descending order of intensity, with percentage fluorescence compared to that of tyrosine, they were tyrosine (100%), phenylalanine (88%), arginine (77.5%), methionine (70.8%), lysine (66.3%), glutamine (61.5%), histidine (51.4%), threonine (50.6%), isoleucine (45.4%), valine (36.9%), leucine (30.7%), asparagine (28.2%), glutamate (22.5%), glycine (22.1%), cysteine (15.2%), serine (13.0%), tryptophan (4.1%), alanine (3.7%), and aspartate (3.2%). Proline, a secondary amine, did not fluoresce. We then compared the fluorescence of a serial dilution of the P. menispermifolia extrafloral nectar mimic to that of phenylalanine solution of equal concentration and to an alanine solution of equal concentration (Fig. 2B). If we had used a phenylalanine solution as the standard, we would have overestimated the amino acid concentration of the P. menispermifolia nectar. Specifically, a phenylalanine concentration of 3 [micro]mol/mL would yield a fluorescence of 51.6; this fluorescence value on the P. menispermifolia curve corresponds to a concentration of 3.24 [micro]mol/mL - an overestimate of 8%. In contrast, an alanine standard would have underestimated the amino acid concentration of P. menispermifolia nectar. Specifically, an alanine concentration of 3 [micro]mol/mL would yield a fluorescence of 5.5; this fluorescence value on the P. menispermifolia curve corresponds to a concentration of 0.49 [micro]mol/mL - an underestimate of 84%.

Discussion

Fluorescamine can be used to estimate the total concentration of amino acids in plant nectars even in small volumes of nectar. We measured concentrations as low as 0.4 [micro]mol/mL in volumes as low as 5 [micro]L. Nectars high in amino acid concentration, like extrafloral nectars and butterfly-pollinated flowers, need to be diluted in order to avoid quenching. Thus, it is possible to survey the total amino acid concentration of the nectars ranging from floral nectars to extrafloral nectars.

The presence of sugar did not affect the fluorescence of a sample. Therefore, sugars need not be included in the solutions used to prepare the standard curve.

The non-fluorescence of secondary amines (e.g., pro-line) leads to an underestimation of amino acid concentrations, similar to the error in ninhydrin tests. In the future, a method could be developed that converts secondary amines to primary amines using sodium hypochlorite and then measures the concentration of the newly-produced primary amines with fluorescamine (Ishida et al. 1981). However, the nectar volume would have to be large enough to split, so that the concentrations of the primary and secondary amines could be measured separately.

Because different primary amines fluoresce at different intensities, fluorescamine provides only an approximation of amino acid concentration. In our test, using phenylalanine for the standard curve would introduce an error of only 8%; using alanine for the standard curve, however, would introduce an error of 84%. Because most nectars contain amino acids of varying fluorescent intensities, the error would be considerably less than in the extreme alanine example. We therefore recommend making a standard curve with an amino acid that fluoresces at moderate intensity, e.g., histidine, threonine, or isoleucine. The error introduced by differential fluorescence can be reduced substantially if one nectar sample is analyzed via HPLC or some other complex procedure. Then, a nectar mimic could be prepared to construct the standard curve and fluorescence measurements of additional nectar samples would be more accurate.

Differences in total fluorescence arising from differential amino acid fluorescence do not affect the use of fluorescamine for within-species comparisons. Because the presence or absence of amino acids remains constant within a species (Baker and Baker 1977, Smith et al. 1990), individual nectar samples will have relatively similar proportions of intensely-, moderately-, and weakly-fluorescing amino acids. Comparisons among species are also possible but will not be as accurate.

The fluorescamine method has several advantages for estimating total amino acid content. It is simpler, faster, and requires less equipment than TLC of dansylated amino acids, HPLC, and amino acid analyzers. It provides a more precise estimate than does ninhydrin. In addition, small volumes can be analyzed easily.

In conclusion, this method bears promise for two kinds of studies: (1) broad surveys, involving between-species comparisons. Such surveys can be used to identify species that warrant further study. For example, an investigator may wish to compare butterfly visitation rates to flowers with similar morphology but different amino acid concentrations, or to flowers with similar amino acid concentrations but different morphologies. This method would allow the investigator to decide which nectars should be analyzed in more detail (i.e., measurement of concentrations of individual amino acids) using more complex and expensive methodologies. (2) more detailed, within-species comparisons. These data can be used to compare plants growing in different environments, experiencing different experimental conditions, or varying in genetic makeup.

Acknowledgments: This work was supported by National Science Foundation REU grant number BIO-9200506 to J. Lanza. We thank the Chemistry Department at State University of New York Fredonia for help and use of their equipment, Anne Zeeh for advice and patience, and Suzanne Swift and Barbara Parsons for follow-up help at University of Arkansas at Little Rock. Barbara Parsons and C. Cheng Kao also provided insightful editorial comments. Finally, for moral support and late-night assistance, we thank Dennis DaSilva, Suzanne Swift, and Jennifer Woods.

Literature Cited

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Potter, C. F., and R. I. Bertin. 1988. Amino acids in artificial nectar: feeding preferences of the flesh fly Sarcophaga bullata. American Midland Naturalist 120:156-162.

Rathman, E. S., J. Lanza, and J. Wilson. 1990. Feeding preferences of flesh flies (Sarcophaga bullata) for sugar-only vs. sugar-amino acid nectars. American Midland Naturalist 124:379-389.

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Smith, L. L., J. Lanza, and G. C. Smith. 1990. Amino acid concentrations in extrafloral nectar of Impatiens sultani increase after simulated herbivory. Ecology 71:107-115.

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Weigele, M., S. L. DeBernardo, J. P. Tengi, and W. Leimgruber. 1972. A novel reagent for the fluorometric assay of primary amines. Journal of the American Chemical Society 94:5927-5928.
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Author:O'Reilly, Erin; Lanza, Janet
Publication:Ecology
Date:Dec 1, 1995
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