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A Fluorescamine Assay for Membrane Protein and Peptide Samples with Non-Amino-Containing Lipids
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
248, 195–201 (1997)
AB972137
A Fluorescamine Assay for Membrane Protein and Peptide Samples with Non-Amino-Containing Lipids L. A. Chung1 Biochemistry Department, University of Virginia School of Medicine, Charlottesville, Virginia 22908
Received November 20, 1996
A method is described for determining the concentration of membrane proteins and peptides in the presence of non-amino-containing lipids. The assay is quantitative when used for purified proteins and peptides of known sequence and qualitative when sequences are unknown or samples contain contaminating proteins. In this method, proteins and peptides are hydrolyzed to amino acids followed by derivatization by fluorescamine and spectroscopic detection in a mixed solvent system. A liquid-phase acid hydrolysis separates lipid from the sample and increases the sensitivity and accuracy of the assay. The aqueous–organic solvent, composed of 40% dimethylformamide, has two advantages. First, it suppresses differences in fluorescence between samples with and without residual hydrolyzed lipid, allowing direct comparison of samples and standards regardless of lipid content. Second, the solvent enhances the fluorescence of amino acid derivatives. While the fluorescence intensities of fluorescamine derivatives reach a maximum at approximately 40% dimethylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations of organic solvent. The selection of an acid hydrolysis mixture based on the fluorescence quenching by different acid mixtures is also reported. q 1997 Academic Press
Methods to determine protein concentrations can be grouped into five general classes: direct protein absorbance, chemical modification of specific residues, biuret assays, dye-binding assays, and protein hydrolysis followed by amino acid quantitation. Protein absorbance methods (reviewed in 1) exploit the ultraviolet absorbance by proteins and rely on estimates of protein extinction coefficients for accuracy (2, 3). Chemical modification methods rely on reactions at specific 1 Address correspondence to the author at Univ. of Virginia Biochemistry Dept., Box 440, Jordan Hall, Rm. 6-17, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Fax: 804-924-5069. E-mail: [email protected].
amino acids such as the reaction of tryptophan with 4(dimethylamino)benaldehyde (4). Biuret assays such as the Lowery are based on the reaction of peptide bonds with copper (5, 6), while dye-binding assays such as the Bradford depend on changes in absorbance when chromophores interact noncovalently with proteins (7, 8). These methods are relative measures that compare the spectroscopic results of a protein sample to those obtained for a standard protein or proteins. Because these assays are optimally selective for certain amino acids, differences between protein structures and amino acid sequences will influence their accuracy (9, 10). The last group of assays, hydrolysis of proteins, degrades proteins into their constituent amino acids (11, 12). Because hydrolysis methods analyze amino acids rather than whole proteins, accurate standards are easily obtained and not complicated by differing protein structures or sequence requirements. For samples of membrane proteins or peptides, the removal of lipid must be considered. Lipids interfere with protein assays, requiring a separation step in the analysis of membrane samples. Previously reported methods for separation include extraction (13), protein precipitation and/or adsorption onto filters (14–17), and chromatography (18, 19). The assay reported here combines a liquid-phase acid hydrolysis with fluorescamine detection (20–22) using a solvent system containing 40% dimethylformamide. Liquid-phase hydrolysis, rather than the more commonly employed gas phase, separates much of the lipid from the sample in this step. The organic solvent suppresses the effects of residual lipid remaining in samples and increases the sensitivity of the assay by enhancing fluorescence intensities. Because of the enhanced fluorescence, shorter excitation path lengths can be used. This decreases quenching due to inner filter effects, resulting in a larger linear range for the assay. While the assay is not suitable for samples containing large amounts of amino lipids such as phosphatidylethanolamine or phosphatidylserine, the possibility of assaying samples with small amounts of amino lipid is discussed. 195
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
Materials. Lipids used in assays were purchased from Avanti Polar Lipids, Alabaster, AL; fluorescamine was purchased from Molecular Probes, Inc. (Eugene, OR); Baker ‘‘Instra-analyzed’’ reagent grade HCl was purchased from J. T. Baker (Phillipsburg, NJ); propionic acid was purchased from Aldrich Chemical Co. (Milwaukee, WI); semiconductor grade NaOH,2 99.99% purity, was obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and solvents were obtained from Sigma Chemical Co. Acid hydrolysis. Culture tubes (13 1 100 mm) with Teflon-lined screwcaps were used for the acid hydrolysis of samples. Either 50- or 100-ml aliquots of sample were brought to a total final volume of 0.5 or 1.0 ml, respectively, with acid mixtures of 1:1 (v/v) propionic acid:concentrated HCl (23), 4 N methanesulfonic acid in propionic acid (24), or 3 N mercaptoethanesulfonic acid in propionic acid (25). For quantitation of peptide concentrations, standard curves were prepared using a 10 mM amino acid standard composed of the same mole ratios of amino acid as the peptide being analyzed. Aliquots of the standards were brought to the same volume as the samples by addition of water or buffer. Samples consisted of buffer only (background tubes), peptide, amino acid standards, lipid, or some combination of these. After addition of acid, the tubes were flushed with N2 gas and covered with Teflon tape before being capped tightly, vortexed, and placed in a heating block set to the appropriate temperature. Samples were normally incubated for 15 to 20 min at 1607C incubation (23), although increasing the incubation temperature to 1807C or the incubation time to 1 h did not affect results. After hydrolysis, tubes were removed from the heating block, cooled slightly, and incubated at 0207C for 0.5 h. Much of the lipid in the sample precipitated out of solution at this point, forming a waxy layer on top of the acid. Occasionally, when the hydrolyzed lipid solidified but remained as a suspension in the acid, samples were spun in a tabletop centrifuge for 5–10 min before chilling. While care was taken to minimize the amount of wax transferred when removing aliquots for further analysis, small amounts of hydrolyzed lipid had no effect on the assay results. It was sometimes necessary to keep the culture tubes on ice while sampling to keep the hydrolyzed lipid solidified. After heating, the acid samples can be stored overnight at 0207C. Minor increases in degradation of amino acids are seen with longer storage times. 2 Abbreviations used: NaOH, sodium hydroxide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DAPC, 1,2-diarachidoyl-snglycero-3-phosphocholine; SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine; SLPC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; SLLPC, 1-stearoyl-2-linolenoyl-sn-glycero-3-phosphocholine.
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Fluorescamine derivatization. Aliquots of 50 ml acid were transferred to 13 1 100-mm test tubes and neutralized using a 10 M NaOH solution made from high purity (99.99%) semiconductor grade NaOH. The amount of NaOH required varied {2–3 ml for individual samples and therefore separate aliquots were tested for neutrality using litmus paper. It was preferable for the samples to test neutral to basic (pH 7.5– 9.0) rather than acidic (pH £6.5). Borate buffer, approximately 0.4 M, pH 9.0, was added to a total sample volume of 0.3 ml and then 0.1 ml of 0.3 mg/ml fluorescamine in acetone was introduced while the solution was vortexing rapidly. Using higher concentrations of borate buffer (0.5 M) resulted in precipitation when dimethylformamide was added to the samples. To increase the concentration range of the assay, fluorescamine solutions of 0.6 and 1.2 mg/ml were also tested (see Results). Very rapid mixing is essential at this point. After the samples were vortexed, they were incubated for 30 min at room temperature before a 50-ml aliquot of 0.1 M ammonium bicarbonate buffer, pH 9.0, was added (200 ml for higher concentrations of fluorescamine) and the tubes were incubated an additional 30 min. Glass-distilled, deionized water and DMF were added to a final volume of 4 ml and the fluorescence of samples was measured. Analysis of residual fatty acid from hydrolysis of POPC. Samples containing 1, 2, 5, or 10 mg POPC were hydrolyzed in 0.5 ml acid according to the procedure described above. Aliquots of 0.1 ml were then dried down under N2 gas and methylated using a diazomethane procedure (26). After methylation, samples were dried and redissolved in 20 ml of chloroform, and 1-ml aliquots were injected into a Hewlett Packard 5890 series II gas chromatograph with a capillary column of cross-linked methylsiloxane (5 m) at 1807C oven temperature. Integrated peak areas and positions were compared with methylated palmitic (C16:0) and oleic (C18:1) acid standards in chloroform to confirm identities and estimate amounts. Since derivatization of the oleic double bond during the acid hydrolysis is likely, only results for palmitic acid are reported. Fluorescence spectroscopy. Fluorescence measurements were obtained using an SLM 8000 fluorimeter and 1 1 0.4-cm path length quartz cuvettes. Incoming and outgoing slits were set at 8 and 2 mm, respectively. For excitation and emission spectra, increments of 1 nm with 1 s averaged readings were used and the results of three spectra were averaged. When higher concentrations of fluorescamine and amino acid or peptide were in the sample, the voltage to the photomultiplier was lowered to prevent detector saturation. Kinetic measurements for the intensity decrease at 475 nm of fluorescamine–alanine derivatives under constant illumination at 397 nm were obtained by integrating intensities over 4 s every 900 s for a period of 36 h (Fig. 9) or every 30 s over a period of 2 h (Fig. 9, inset).
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citation or emission spectra for samples with and without acid mixtures (data not shown). The similarity of excitation and emission maximum wavelengths and overall spectra implies that quenching was not due to a covalent modification of the amino acid or its fluorescamine derivative by the acid mixtures. Based on the fluorescence quenching of the different acid mixtures, only the propionic acid:HCl mixture was used in further development of the assay. It should be emphasized that neutralization of the acid mixture with NaOH is the most problematic step in the assay. Failure to form fluorescamine derivatives was invariably due to incorrect pH of the samples. It was necessary to determine the amount of 10 M NaOH required for neutralization to {0.5 ml for each sample, normally 54–61 ml of 10 M NaOH for 50 ml of acid. While samples of the same composition required similar volumes of NaOH, different preparations or bottles of NaOH and acids could change required amounts by as much as 5 ml. The acid hydrolysis volumes of 0.5 to 1 ml allowed the testing of individual samples using litmus paper before preparing the actual assay tubes. FIG. 1. Effect of acid mixtures and amount of acid on the fluorescence of standard curves. The fluorescence intensities of fluorescamine–alanine derivatives with (A) 1:1 concentrated HCl: propionic acid, and (B) propionic acid containing 4 N methanesulfonic acid. The symbols represent measurements with no acid present (j), with 50 ml of acid added and neutralized (s) and with 100 ml of acid (n).
RESULTS
Effect of acid hydrolysis mixtures on fluorescence. Figure 1 shows the quenching effect of the neutralized acid mixtures on the fluorescence intensity of a fluorescamine –amino acid derivative. Three acid hydrolysis mixtures were tested in this report—1:1 (v/v) propionic acid:concentrated HCl (23), propionic acid containing 4 N methanesulfonic acid (24), and propionic acid containing 3 N mercaptoethanesulfonic acid (25). Fluorescamine– alanine derivatives were used to generate standard curves with no acid (solid squares), with a 50- or 100-ml aliquot of the 1:1 (v/v) propionic acid:concentrated HCl mixture (Fig. 1A), and with a 50- or 100-ml aliquot of propionic acid containing 4 N methanesulfonic acid (Fig. 1B). The mixture of propionic acid containing 3 N mercaptoethanesulfonic acid gave results similar to 4 N methanesulfonic acid (not shown). As described under Materials and Methods, the acid mixture was added to the amino acid standard, heated, and then neutralized with 10 N NaOH before forming the fluorescamine derivative. All the acid mixtures resulted in some quenching of fluorescence, the least severe being the 50-ml addition of 1:1 propionic acid:HCl. Fluorescence quenching was greater for the sulfonic acids than for acids that did not contain sulfur groups. Aside from the decrease in fluorescence intensities, no change was seen in the ex-
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Effect of lipid on fluorescence. While much of the hydrolyzed lipid solidifies out as a waxy substance in the acid hydrolysis mixture, some remains in solution. When samples of 1, 2, 5, and 10 mg POPC/0.5 ml hydrolysis acid were analyzed for residual fatty acid (see Materials and Methods), a final concentration of approximately 1.6 mM palmitic acid in the hydrolysis acid was determined for all samples. Figures 2 and 3 show how the residual hydrolyzed POPC affects the fluorescent properties of fluorescamine–alanine derivatives, both decreasing the fluorescence intensity (Fig. 2A) and blueshifting the maximum emission wavelength (Fig. 3A) in a buffer-only solvent system. The basis for these effects is necessarily the interaction of fluorophore with residual hydrolyzed lipid in the aqueous sample since that is the only difference between samples. Altered fluorescence properties are a problem since quantitation of protein or peptide requires comparison of lipid-containing samples with lipid-free samples and standard curves. Testing of various protocols designed to suppress differences between lipid and lipid-free samples included screening different detergents and solvents such as alcohols and ketones. The mixed solvent system described below was found to be the simplest solution. Effect of dimethylformamide on fluorescence. No difference in fluorescence is seen between samples with and without lipid in the presence of 40% (v/v) dimethylformamide. This is illustrated in Figs. 2B and 3B; both the fluorescence intensities (Fig. 2) and excitation and emission spectra (Fig. 3) remain the same regardless of the presence of hydrolyzed lipid. The suppression of fluorescence differences caused by the presence of hydrolyzed lipid does not depend on the amino acid residue or the lipid acyl chain. While
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FIG. 2. Effect of residual hydrolyzed lipid on the fluorescence of standard curves in (A) aqueous buffer and (B) buffer plus 40% dimethylformamide. Acid hydrolysis mixtures containing 5–10 mg of POPC and varying amounts of alanine were prepared and 50-ml aliquots were used for derivatization with fluorescamine (see Materials and Methods for details). Symbols represent the presence (n) or absence (l) of lipid in hydrolysis samples.
the spectra and standard curves shown were generated using alanine, the same result was obtained for leucine, glycine, serine, tyrosine, and aspartic acids (not shown). It was found that regardless of the polarity of the residue side chain, no appreciable difference in fluorescence properties was detected between samples with and without lipid when 40% DMF was included. The effects of changing the lipid acyl chain length or the unsaturation of acyl chains were investigated. The amount of hydrolyzed lipid remaining in solution and its interaction with the fluorescamine–amino acid derivatives depends in part on the chemical structure of the lipid. POPC, which contains one saturated C16 acyl chain (1-acyl position) and one C18 acyl chain containing a single double bond (2-acyl position), was the lipid used for developing and testing this assay. However, saturated lipids having acyl chain lengths of C14 (dimyristoyl, DMPC), C16 (dipalmitoyl, DPPC), C18 (distearoyl, DSPC), and C20 (diarachidoyl, DAPC) carbons and di-C18 lipids with one chain containing 0 (distearoyl, DSPC), 1 (stearoyl-oleoyl, SOPC), 2 (stearoyl-linoleoyl, SLPC), or 3 (stearoyl-linolenoyl, SLLPC) double bonds were tested and compared. The different lipid acyl chains gave the same results as POPC (not shown), although some required longer incubation times at 0207C to solidify out of solution. The effect of varying the amount of DMF on fluores-
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cence was also studied. Figures 4 and 5 show the change in fluorescence intensities and Fig. 6 shows the changes in maximum emission wavelengths when the percentage of dimethylformamide in the solvent is varied. The excitation spectra were unaffected by the amount of DMF present (not shown). A steady blueshift in the emission wavelength is seen with increasing percentages of DMF in the solvent (Fig. 6), an effect characteristic of an increasingly hydrophobic environment. Unexpectedly, the fluorescent intensities have a maximum at approximately 40% DMF and this maximum is not dependent on either the type of amino acid residue or its concentration (Figs. 4 and 5). While halving the concentration of amino acid in the samples decreases the fluorescence intensities, the relative maximum intensities at 40% DMF remains unchanged. A similar effect on fluorescamine derivatives has been noted for DMSO-containing solvents (27). Linear range of fluorescamine derivatives in DMF. The linear range as a function of the fluorescamine concentration was determined for different amino acids in 40% (v/v) DMF. Using the experimental conditions described (see Materials and Methods), fluorescence intensities were linear through 50 nmol/ml of fluorescamine–amino acid derivative. The difference between this range and smaller ranges reported previously (20, 28) is based partly on the cuvette geometry. A short
FIG. 3. Effect of residual hydrolyzed lipid on excitation and emission spectra for fluorescamine–alanine derivatives in (A) aqueous buffer and (B) buffer plus 40% dimethylformamide. The fluorescence spectra of 20 nmol samples from Fig. 2 without lipid (---) or containing hydrolyzed lipid (—). See Materials and Methods for details of sample preparation and fluorescence spectroscopy.
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FIG. 4. Effect of varying dimethylformamide concentrations on the fluorescence intensities at maximum emission wavelengths for different fluorescamine–amino acid derivatives. Filled symbols indicate samples containing 10 nmol of amino acid, hollow symbols indicate samples with 20 nmol amino acid. The amino acids tested were alanine (j), serine (l), aspartic acid (m), lysine (L), and leucine (,).
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FIG. 6. Effect of varying dimethylformamide concentrations on the emission wavelength maximums of different fluorescamine–amino acid derivatives. Samples contained 20 nmol of the following amino acids: alanine (h), serine (s), aspartic acid (n), lysine (L), and leucine (,).
excitation path length combined with a wide excitation slit ensures proper irradiation of the sample and decreases quenching due to inner filter effects. Figure 7 compares the standard curves of lysine and alanine when 200 ml of 0.3, 0.6, or 1.2 mg/ml fluorescamine in acetone is used to form the derivatives in the assay. The absolute fluorescent intensities shown in Fig. 7 differ from previous figures because of the lower voltages used to prevent detector saturation at the higher concentrations of amino acid. The extent of the linear range increases from 50 nmol of amino acid in a 4-ml sample when using a 0.3 mg fluorescamine/ ml stock solution (Fig. 7, hollow squares) to 200 nmol of amino acid when the concentration of fluorescamine is 1.2 mg/ml (Fig. 7, hollow circles). As expected, the deviation from linearity is greater for lysine (Fig. 7A) than alanine (Fig. 7B) due to reagent consumption by the lysine e-amino group. However, both amino acids are linear in the 0 to 200 nM range when the higher concentrations of fluorescamine are used. DISCUSSION
FIG. 5. Effect of varying dimethylformamide concentrations on the fluorescence intensities at 475 nm for different fluorescamine–amino acid derivatives. Filled symbols indicate samples containing 10 nmol of amino acid, hollow symbols indicate samples with 20 nmol amino acid. The amino acids tested were alanine (j, h), serine (l, s), aspartic acid (m, n), lysine (L), and leucine (,).
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The concentrated HCl:propionic acid mixture used in this assay has certain advantages over other hydrolysis mixtures reported in the literature. This mixture showed the lowest quenching of fluorophore (Fig. 1), required the shortest heat incubation times (15–20 min vs §24 h), and readily hydrolyzed peptides containing hydrophobic residues, which can be recalcitrant to stan-
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FIG. 7. Effect of fluorescamine concentration on the linear range of amino acid derivatives in buffers containing 40% dimethylformamide. Standard curves for (A) fluorescamine–lysine and (B) fluorescamine– alanine derivatives prepared using 200 ml of fluorescamine in acetone stock solutions according to procedures outlined under Materials and Methods. The concentrations of fluorescamine stock solutions used were 0.3 mg fluorescamine/ml acetone (h), 0.6 mg fluorescamine/ml acetone (s), and 1.2 mg fluorescamine/ml acetone (n).
dard hydrolysis protocols (29, 30; Fig. 8). This assay has been routinely used to analyze peptide samples containing up to 0.3 M POPC (29, 30). Using a liquid-phase hydrolysis, rather than the usual gas phase, separates most of the hydrolyzed lipid from the sample without requiring an additional extraction step. In a gas-phase hydrolysis, dried samples are exposed to acid vapor at elevated temperatures. By drying samples in the presence of lipid, hydrophobic sequences can form complexes with lipid, protecting sequences from the gaseous acid and preventing hydrolysis. Incomplete hydrolysis of peptide sequences was not a problem using this liquidphase protocol regardless of lipid content. While most of the hydrolyzed lipid solidifies and separates out, enough remains in solution to result in differences between lipid-containing and lipid-free samples (Figs. 2 and 3). Using a mixed solvent system for the fluorescence measurements eliminated the difference between these samples. Dimethylformamide was chosen over other possible additives for both ease in sample handling and spectroscopic advantages. Detergents will foam and some have high background fluorescence; alcohols and ketones can evaporate, changing the concentration of the sample during measurements. The mixed solvent results in an enhanced fluorescence signal, increasing the sensitivity of the measurements.
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This allowed the use of shorter path lengths to avoid inner filter effects, extending the linear range over those reported previously (20, 28). The reproducibility of the assay is good, especially when standard curves are included for comparison. The error bars shown in Fig. 8 for melittin are representative of the deviations commonly observed for other peptide sequences and standards with or without lipid present. In common with other analytical assays, certain precautions will improve both accuracy and precision. Using Hamilton syringes, rather that air-displacement pipets, to measure and transfer samples for acid hydrolysis markedly decreases the deviations due to volumetric error. The various amino acid derivatives differ in their fluorescence intensities (22; Figs. 4 and 5) and standards with the same amino acid composition as the proteins or peptides being quantitated are advised for the greatest accuracy. Acid hydrolysis and neutralization should be carried out on standards as well as samples. The fluorescamine derivatives are stable in 40% DMF at room temperature; little to no change in intensity was seen over a period of a week. The fluorescence intensities do degrade with constant illumination at excitation wavelengths (Fig. 9) but exposure to room lighting for a week had no noticeable effect. The sensitivity of fluorescamine-based assays has
FIG. 8. Variability of assay results for the hydrolysis of melittin. Single samples of a 2 mg/ml stock melittin solution in deionized water (86% pure, Sigma Chemical Co.) were diluted to different concentrations and then hydrolyzed according to procedures outlined under Materials and Methods. Triplicate aliquots from the acid hydrolysis were then neutralized and derivatized with fluorescamine, and the fluorescence was measured in 40% DMF. Error bars indicate the standard deviation of these triplicate measurements. The linear regression on the intensity values (solid line) gives an R value of 0.9985.
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ACKNOWLEDGMENTS The author gratefully acknowledges Professor Thomas E. Thompson for his encouragement and support of this work and reading of the manuscript. This work was funded by NIH Grant GM-18628 to Professor T. E. Thompson.
REFERENCES
FIG. 9. Photodegradation of fluorescamine–alanine under constant illumination at 397 nm. Samples contained 20–30 nmol of fluorescamine–alanine. See Materials and Methods for details of sample preparation and spectroscopy.
been explored by other authors (20); this assay is based in part on the results of this previous work. As expected, the sensitivity of the assay is altered by substances that compete for fluorescamine reagent and increase the background fluorescence. However, the assay can reliably detect 2 nmol of amino acid in 4-ml assay volumes when the initial samples contain 0.1 M Mops or Hepes buffer and 0.3 M POPC. While other common substances containing reactive amino groups, such as nucleotides, nucleosides, and Tris buffer, were not tested, potential interference due to reagent consumption should be eliminated by using higher concentrations of fluorescamine stock solutions. Attempts to modify the assay for use with the amino-containing lipids phosphatidylethanolamine and phosphatidylserine were unsuccessful. The amino groups on both lipids can be blocked by acetylation or succinylation, but the blocking groups are unstable to acid hydrolysis. As discussed above, these lipids will interfere with the assay both by reducing the amount of fluorescamine available to react with amino acid and by contributing a large background fluorescence. It may be possible to correct for small amounts of these lipids by using high concentrations of fluorescamine solutions to form derivatives and estimating the lipid fluorescence contribution using separate samples containing only lipid, but this was not tested in this report.
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1. Stoscheck, C. M. (1990) in Methods in Enzymology (Deutscher, M. P., Ed.), Vol. 182, pp. 50–68, Academic Press, New York. 2. Mach, H., Middaugh, C. R., and Lewis, R. V. (1992) Anal. Biochem. 200, 74–80. 3. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319–326. 4. Urbaneja, M.-A., Alonso, A., and Gon˜i, F. M. (1985) J. Biochem. Biophys. Methods 11, 341–345. 5. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76–85. 6. Lowery, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 7. Greenberg, C. S., and Craddock, P. R. (1982) Clin. Chem. 28, 1725–1726. 8. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 9. Compton, S. J., and Jones, C. G. (1985) Anal. Biochem. 151, 369–374. 10. Chou, S., and Goldstein, A. (1960) Biochem. J. 75, 109–115. 11. Horstmann, H.-J. (1979) Anal. Biochem. 96, 130–138. 12. Butcher, E. C., and Lowry, O. H. (1976) Anal. Biochem. 76, 502–523. 13. Tornqvist, H., and Belfrage, P. (1976) J. Lipid Res. 17, 542–545. 14. Minamide, L. S., and Bamburg, J. R. (1990) Anal. Biochem. 190, 66–70. 15. Brown, R. E., Jarvis, K. L., and Hyland, K. J. (1989) Anal. Biochem. 180, 136–139. 16. Kaplan, R. S., and Pedersen, P. L. (1985) Anal. Biochem. 150, 97–104. 17. Nakamura, H., and Pisano, J. J. (1976) Arch. Biochem. Biophys. 172, 102–105. 18. Wimley, W. C., and White, S. H. (1993) 213, 213–217. 19. Schiltz, E., Schnackerz, K. D., and Gracy, R. W. (1977) Anal. Biochem. 79, 33–41. 20. Castell, J. V., Cervera, M., and Marco, R. (1979) Anal. Biochem. 99, 379–391. 21. Bo¨hlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213–220. 22. Udenfriend, S., Stein, S., Bo¨hlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 871–872. 23. Westall, F., and Hesser, H. (1974) Anal. Biochem. 61, 610–613. 24. Simpson, R. J., Neuberger, M. R., and Liu, T.-Y. (1976) J. Biol. Chem. 251, 1936–1940. 25. Penke, B., Ferenczi, R., and Kova´cs, K. (1974) Anal. Biochem. 60, 45–50. 26. Christie, W. B. (1973) Lipid Analysis, Pergamon Press, Oxford, England. 27. Froehlich, P. M., and Cunningham, T. D. (1976) Anal. Chem. Acta 84, 427–430. 28. de Bernardo, S., Weigele, M., Tome, V., Manhart, K., and Leimgurber, W. (1974) Arch. Biochem. Biophys. 163, 390–399. 29. Chung, L. A., and Thompson, T. E. (1996) Biochemistry 35, 11343–11354. 30. Chung, L. A., and Thompson, T. E. (1997) Manuscript in preparation.
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AB972137
A Fluorescamine Assay for Membrane Protein and Peptide Samples with Non-Amino-Containing Lipids L. A. Chung1 Biochemistry Department, University of Virginia School of Medicine, Charlottesville, Virginia 22908
Received November 20, 1996
A method is described for determining the concentration of membrane proteins and peptides in the presence of non-amino-containing lipids. The assay is quantitative when used for purified proteins and peptides of known sequence and qualitative when sequences are unknown or samples contain contaminating proteins. In this method, proteins and peptides are hydrolyzed to amino acids followed by derivatization by fluorescamine and spectroscopic detection in a mixed solvent system. A liquid-phase acid hydrolysis separates lipid from the sample and increases the sensitivity and accuracy of the assay. The aqueous–organic solvent, composed of 40% dimethylformamide, has two advantages. First, it suppresses differences in fluorescence between samples with and without residual hydrolyzed lipid, allowing direct comparison of samples and standards regardless of lipid content. Second, the solvent enhances the fluorescence of amino acid derivatives. While the fluorescence intensities of fluorescamine derivatives reach a maximum at approximately 40% dimethylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations of organic solvent. The selection of an acid hydrolysis mixture based on the fluorescence quenching by different acid mixtures is also reported. q 1997 Academic Press
Methods to determine protein concentrations can be grouped into five general classes: direct protein absorbance, chemical modification of specific residues, biuret assays, dye-binding assays, and protein hydrolysis followed by amino acid quantitation. Protein absorbance methods (reviewed in 1) exploit the ultraviolet absorbance by proteins and rely on estimates of protein extinction coefficients for accuracy (2, 3). Chemical modification methods rely on reactions at specific 1 Address correspondence to the author at Univ. of Virginia Biochemistry Dept., Box 440, Jordan Hall, Rm. 6-17, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Fax: 804-924-5069. E-mail: [email protected].
amino acids such as the reaction of tryptophan with 4(dimethylamino)benaldehyde (4). Biuret assays such as the Lowery are based on the reaction of peptide bonds with copper (5, 6), while dye-binding assays such as the Bradford depend on changes in absorbance when chromophores interact noncovalently with proteins (7, 8). These methods are relative measures that compare the spectroscopic results of a protein sample to those obtained for a standard protein or proteins. Because these assays are optimally selective for certain amino acids, differences between protein structures and amino acid sequences will influence their accuracy (9, 10). The last group of assays, hydrolysis of proteins, degrades proteins into their constituent amino acids (11, 12). Because hydrolysis methods analyze amino acids rather than whole proteins, accurate standards are easily obtained and not complicated by differing protein structures or sequence requirements. For samples of membrane proteins or peptides, the removal of lipid must be considered. Lipids interfere with protein assays, requiring a separation step in the analysis of membrane samples. Previously reported methods for separation include extraction (13), protein precipitation and/or adsorption onto filters (14–17), and chromatography (18, 19). The assay reported here combines a liquid-phase acid hydrolysis with fluorescamine detection (20–22) using a solvent system containing 40% dimethylformamide. Liquid-phase hydrolysis, rather than the more commonly employed gas phase, separates much of the lipid from the sample in this step. The organic solvent suppresses the effects of residual lipid remaining in samples and increases the sensitivity of the assay by enhancing fluorescence intensities. Because of the enhanced fluorescence, shorter excitation path lengths can be used. This decreases quenching due to inner filter effects, resulting in a larger linear range for the assay. While the assay is not suitable for samples containing large amounts of amino lipids such as phosphatidylethanolamine or phosphatidylserine, the possibility of assaying samples with small amounts of amino lipid is discussed. 195
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
Materials. Lipids used in assays were purchased from Avanti Polar Lipids, Alabaster, AL; fluorescamine was purchased from Molecular Probes, Inc. (Eugene, OR); Baker ‘‘Instra-analyzed’’ reagent grade HCl was purchased from J. T. Baker (Phillipsburg, NJ); propionic acid was purchased from Aldrich Chemical Co. (Milwaukee, WI); semiconductor grade NaOH,2 99.99% purity, was obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and solvents were obtained from Sigma Chemical Co. Acid hydrolysis. Culture tubes (13 1 100 mm) with Teflon-lined screwcaps were used for the acid hydrolysis of samples. Either 50- or 100-ml aliquots of sample were brought to a total final volume of 0.5 or 1.0 ml, respectively, with acid mixtures of 1:1 (v/v) propionic acid:concentrated HCl (23), 4 N methanesulfonic acid in propionic acid (24), or 3 N mercaptoethanesulfonic acid in propionic acid (25). For quantitation of peptide concentrations, standard curves were prepared using a 10 mM amino acid standard composed of the same mole ratios of amino acid as the peptide being analyzed. Aliquots of the standards were brought to the same volume as the samples by addition of water or buffer. Samples consisted of buffer only (background tubes), peptide, amino acid standards, lipid, or some combination of these. After addition of acid, the tubes were flushed with N2 gas and covered with Teflon tape before being capped tightly, vortexed, and placed in a heating block set to the appropriate temperature. Samples were normally incubated for 15 to 20 min at 1607C incubation (23), although increasing the incubation temperature to 1807C or the incubation time to 1 h did not affect results. After hydrolysis, tubes were removed from the heating block, cooled slightly, and incubated at 0207C for 0.5 h. Much of the lipid in the sample precipitated out of solution at this point, forming a waxy layer on top of the acid. Occasionally, when the hydrolyzed lipid solidified but remained as a suspension in the acid, samples were spun in a tabletop centrifuge for 5–10 min before chilling. While care was taken to minimize the amount of wax transferred when removing aliquots for further analysis, small amounts of hydrolyzed lipid had no effect on the assay results. It was sometimes necessary to keep the culture tubes on ice while sampling to keep the hydrolyzed lipid solidified. After heating, the acid samples can be stored overnight at 0207C. Minor increases in degradation of amino acids are seen with longer storage times. 2 Abbreviations used: NaOH, sodium hydroxide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DAPC, 1,2-diarachidoyl-snglycero-3-phosphocholine; SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine; SLPC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; SLLPC, 1-stearoyl-2-linolenoyl-sn-glycero-3-phosphocholine.
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Fluorescamine derivatization. Aliquots of 50 ml acid were transferred to 13 1 100-mm test tubes and neutralized using a 10 M NaOH solution made from high purity (99.99%) semiconductor grade NaOH. The amount of NaOH required varied {2–3 ml for individual samples and therefore separate aliquots were tested for neutrality using litmus paper. It was preferable for the samples to test neutral to basic (pH 7.5– 9.0) rather than acidic (pH £6.5). Borate buffer, approximately 0.4 M, pH 9.0, was added to a total sample volume of 0.3 ml and then 0.1 ml of 0.3 mg/ml fluorescamine in acetone was introduced while the solution was vortexing rapidly. Using higher concentrations of borate buffer (0.5 M) resulted in precipitation when dimethylformamide was added to the samples. To increase the concentration range of the assay, fluorescamine solutions of 0.6 and 1.2 mg/ml were also tested (see Results). Very rapid mixing is essential at this point. After the samples were vortexed, they were incubated for 30 min at room temperature before a 50-ml aliquot of 0.1 M ammonium bicarbonate buffer, pH 9.0, was added (200 ml for higher concentrations of fluorescamine) and the tubes were incubated an additional 30 min. Glass-distilled, deionized water and DMF were added to a final volume of 4 ml and the fluorescence of samples was measured. Analysis of residual fatty acid from hydrolysis of POPC. Samples containing 1, 2, 5, or 10 mg POPC were hydrolyzed in 0.5 ml acid according to the procedure described above. Aliquots of 0.1 ml were then dried down under N2 gas and methylated using a diazomethane procedure (26). After methylation, samples were dried and redissolved in 20 ml of chloroform, and 1-ml aliquots were injected into a Hewlett Packard 5890 series II gas chromatograph with a capillary column of cross-linked methylsiloxane (5 m) at 1807C oven temperature. Integrated peak areas and positions were compared with methylated palmitic (C16:0) and oleic (C18:1) acid standards in chloroform to confirm identities and estimate amounts. Since derivatization of the oleic double bond during the acid hydrolysis is likely, only results for palmitic acid are reported. Fluorescence spectroscopy. Fluorescence measurements were obtained using an SLM 8000 fluorimeter and 1 1 0.4-cm path length quartz cuvettes. Incoming and outgoing slits were set at 8 and 2 mm, respectively. For excitation and emission spectra, increments of 1 nm with 1 s averaged readings were used and the results of three spectra were averaged. When higher concentrations of fluorescamine and amino acid or peptide were in the sample, the voltage to the photomultiplier was lowered to prevent detector saturation. Kinetic measurements for the intensity decrease at 475 nm of fluorescamine–alanine derivatives under constant illumination at 397 nm were obtained by integrating intensities over 4 s every 900 s for a period of 36 h (Fig. 9) or every 30 s over a period of 2 h (Fig. 9, inset).
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citation or emission spectra for samples with and without acid mixtures (data not shown). The similarity of excitation and emission maximum wavelengths and overall spectra implies that quenching was not due to a covalent modification of the amino acid or its fluorescamine derivative by the acid mixtures. Based on the fluorescence quenching of the different acid mixtures, only the propionic acid:HCl mixture was used in further development of the assay. It should be emphasized that neutralization of the acid mixture with NaOH is the most problematic step in the assay. Failure to form fluorescamine derivatives was invariably due to incorrect pH of the samples. It was necessary to determine the amount of 10 M NaOH required for neutralization to {0.5 ml for each sample, normally 54–61 ml of 10 M NaOH for 50 ml of acid. While samples of the same composition required similar volumes of NaOH, different preparations or bottles of NaOH and acids could change required amounts by as much as 5 ml. The acid hydrolysis volumes of 0.5 to 1 ml allowed the testing of individual samples using litmus paper before preparing the actual assay tubes. FIG. 1. Effect of acid mixtures and amount of acid on the fluorescence of standard curves. The fluorescence intensities of fluorescamine–alanine derivatives with (A) 1:1 concentrated HCl: propionic acid, and (B) propionic acid containing 4 N methanesulfonic acid. The symbols represent measurements with no acid present (j), with 50 ml of acid added and neutralized (s) and with 100 ml of acid (n).
RESULTS
Effect of acid hydrolysis mixtures on fluorescence. Figure 1 shows the quenching effect of the neutralized acid mixtures on the fluorescence intensity of a fluorescamine –amino acid derivative. Three acid hydrolysis mixtures were tested in this report—1:1 (v/v) propionic acid:concentrated HCl (23), propionic acid containing 4 N methanesulfonic acid (24), and propionic acid containing 3 N mercaptoethanesulfonic acid (25). Fluorescamine– alanine derivatives were used to generate standard curves with no acid (solid squares), with a 50- or 100-ml aliquot of the 1:1 (v/v) propionic acid:concentrated HCl mixture (Fig. 1A), and with a 50- or 100-ml aliquot of propionic acid containing 4 N methanesulfonic acid (Fig. 1B). The mixture of propionic acid containing 3 N mercaptoethanesulfonic acid gave results similar to 4 N methanesulfonic acid (not shown). As described under Materials and Methods, the acid mixture was added to the amino acid standard, heated, and then neutralized with 10 N NaOH before forming the fluorescamine derivative. All the acid mixtures resulted in some quenching of fluorescence, the least severe being the 50-ml addition of 1:1 propionic acid:HCl. Fluorescence quenching was greater for the sulfonic acids than for acids that did not contain sulfur groups. Aside from the decrease in fluorescence intensities, no change was seen in the ex-
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Effect of lipid on fluorescence. While much of the hydrolyzed lipid solidifies out as a waxy substance in the acid hydrolysis mixture, some remains in solution. When samples of 1, 2, 5, and 10 mg POPC/0.5 ml hydrolysis acid were analyzed for residual fatty acid (see Materials and Methods), a final concentration of approximately 1.6 mM palmitic acid in the hydrolysis acid was determined for all samples. Figures 2 and 3 show how the residual hydrolyzed POPC affects the fluorescent properties of fluorescamine–alanine derivatives, both decreasing the fluorescence intensity (Fig. 2A) and blueshifting the maximum emission wavelength (Fig. 3A) in a buffer-only solvent system. The basis for these effects is necessarily the interaction of fluorophore with residual hydrolyzed lipid in the aqueous sample since that is the only difference between samples. Altered fluorescence properties are a problem since quantitation of protein or peptide requires comparison of lipid-containing samples with lipid-free samples and standard curves. Testing of various protocols designed to suppress differences between lipid and lipid-free samples included screening different detergents and solvents such as alcohols and ketones. The mixed solvent system described below was found to be the simplest solution. Effect of dimethylformamide on fluorescence. No difference in fluorescence is seen between samples with and without lipid in the presence of 40% (v/v) dimethylformamide. This is illustrated in Figs. 2B and 3B; both the fluorescence intensities (Fig. 2) and excitation and emission spectra (Fig. 3) remain the same regardless of the presence of hydrolyzed lipid. The suppression of fluorescence differences caused by the presence of hydrolyzed lipid does not depend on the amino acid residue or the lipid acyl chain. While
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FIG. 2. Effect of residual hydrolyzed lipid on the fluorescence of standard curves in (A) aqueous buffer and (B) buffer plus 40% dimethylformamide. Acid hydrolysis mixtures containing 5–10 mg of POPC and varying amounts of alanine were prepared and 50-ml aliquots were used for derivatization with fluorescamine (see Materials and Methods for details). Symbols represent the presence (n) or absence (l) of lipid in hydrolysis samples.
the spectra and standard curves shown were generated using alanine, the same result was obtained for leucine, glycine, serine, tyrosine, and aspartic acids (not shown). It was found that regardless of the polarity of the residue side chain, no appreciable difference in fluorescence properties was detected between samples with and without lipid when 40% DMF was included. The effects of changing the lipid acyl chain length or the unsaturation of acyl chains were investigated. The amount of hydrolyzed lipid remaining in solution and its interaction with the fluorescamine–amino acid derivatives depends in part on the chemical structure of the lipid. POPC, which contains one saturated C16 acyl chain (1-acyl position) and one C18 acyl chain containing a single double bond (2-acyl position), was the lipid used for developing and testing this assay. However, saturated lipids having acyl chain lengths of C14 (dimyristoyl, DMPC), C16 (dipalmitoyl, DPPC), C18 (distearoyl, DSPC), and C20 (diarachidoyl, DAPC) carbons and di-C18 lipids with one chain containing 0 (distearoyl, DSPC), 1 (stearoyl-oleoyl, SOPC), 2 (stearoyl-linoleoyl, SLPC), or 3 (stearoyl-linolenoyl, SLLPC) double bonds were tested and compared. The different lipid acyl chains gave the same results as POPC (not shown), although some required longer incubation times at 0207C to solidify out of solution. The effect of varying the amount of DMF on fluores-
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cence was also studied. Figures 4 and 5 show the change in fluorescence intensities and Fig. 6 shows the changes in maximum emission wavelengths when the percentage of dimethylformamide in the solvent is varied. The excitation spectra were unaffected by the amount of DMF present (not shown). A steady blueshift in the emission wavelength is seen with increasing percentages of DMF in the solvent (Fig. 6), an effect characteristic of an increasingly hydrophobic environment. Unexpectedly, the fluorescent intensities have a maximum at approximately 40% DMF and this maximum is not dependent on either the type of amino acid residue or its concentration (Figs. 4 and 5). While halving the concentration of amino acid in the samples decreases the fluorescence intensities, the relative maximum intensities at 40% DMF remains unchanged. A similar effect on fluorescamine derivatives has been noted for DMSO-containing solvents (27). Linear range of fluorescamine derivatives in DMF. The linear range as a function of the fluorescamine concentration was determined for different amino acids in 40% (v/v) DMF. Using the experimental conditions described (see Materials and Methods), fluorescence intensities were linear through 50 nmol/ml of fluorescamine–amino acid derivative. The difference between this range and smaller ranges reported previously (20, 28) is based partly on the cuvette geometry. A short
FIG. 3. Effect of residual hydrolyzed lipid on excitation and emission spectra for fluorescamine–alanine derivatives in (A) aqueous buffer and (B) buffer plus 40% dimethylformamide. The fluorescence spectra of 20 nmol samples from Fig. 2 without lipid (---) or containing hydrolyzed lipid (—). See Materials and Methods for details of sample preparation and fluorescence spectroscopy.
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FIG. 4. Effect of varying dimethylformamide concentrations on the fluorescence intensities at maximum emission wavelengths for different fluorescamine–amino acid derivatives. Filled symbols indicate samples containing 10 nmol of amino acid, hollow symbols indicate samples with 20 nmol amino acid. The amino acids tested were alanine (j), serine (l), aspartic acid (m), lysine (L), and leucine (,).
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FIG. 6. Effect of varying dimethylformamide concentrations on the emission wavelength maximums of different fluorescamine–amino acid derivatives. Samples contained 20 nmol of the following amino acids: alanine (h), serine (s), aspartic acid (n), lysine (L), and leucine (,).
excitation path length combined with a wide excitation slit ensures proper irradiation of the sample and decreases quenching due to inner filter effects. Figure 7 compares the standard curves of lysine and alanine when 200 ml of 0.3, 0.6, or 1.2 mg/ml fluorescamine in acetone is used to form the derivatives in the assay. The absolute fluorescent intensities shown in Fig. 7 differ from previous figures because of the lower voltages used to prevent detector saturation at the higher concentrations of amino acid. The extent of the linear range increases from 50 nmol of amino acid in a 4-ml sample when using a 0.3 mg fluorescamine/ ml stock solution (Fig. 7, hollow squares) to 200 nmol of amino acid when the concentration of fluorescamine is 1.2 mg/ml (Fig. 7, hollow circles). As expected, the deviation from linearity is greater for lysine (Fig. 7A) than alanine (Fig. 7B) due to reagent consumption by the lysine e-amino group. However, both amino acids are linear in the 0 to 200 nM range when the higher concentrations of fluorescamine are used. DISCUSSION
FIG. 5. Effect of varying dimethylformamide concentrations on the fluorescence intensities at 475 nm for different fluorescamine–amino acid derivatives. Filled symbols indicate samples containing 10 nmol of amino acid, hollow symbols indicate samples with 20 nmol amino acid. The amino acids tested were alanine (j, h), serine (l, s), aspartic acid (m, n), lysine (L), and leucine (,).
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The concentrated HCl:propionic acid mixture used in this assay has certain advantages over other hydrolysis mixtures reported in the literature. This mixture showed the lowest quenching of fluorophore (Fig. 1), required the shortest heat incubation times (15–20 min vs §24 h), and readily hydrolyzed peptides containing hydrophobic residues, which can be recalcitrant to stan-
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FIG. 7. Effect of fluorescamine concentration on the linear range of amino acid derivatives in buffers containing 40% dimethylformamide. Standard curves for (A) fluorescamine–lysine and (B) fluorescamine– alanine derivatives prepared using 200 ml of fluorescamine in acetone stock solutions according to procedures outlined under Materials and Methods. The concentrations of fluorescamine stock solutions used were 0.3 mg fluorescamine/ml acetone (h), 0.6 mg fluorescamine/ml acetone (s), and 1.2 mg fluorescamine/ml acetone (n).
dard hydrolysis protocols (29, 30; Fig. 8). This assay has been routinely used to analyze peptide samples containing up to 0.3 M POPC (29, 30). Using a liquid-phase hydrolysis, rather than the usual gas phase, separates most of the hydrolyzed lipid from the sample without requiring an additional extraction step. In a gas-phase hydrolysis, dried samples are exposed to acid vapor at elevated temperatures. By drying samples in the presence of lipid, hydrophobic sequences can form complexes with lipid, protecting sequences from the gaseous acid and preventing hydrolysis. Incomplete hydrolysis of peptide sequences was not a problem using this liquidphase protocol regardless of lipid content. While most of the hydrolyzed lipid solidifies and separates out, enough remains in solution to result in differences between lipid-containing and lipid-free samples (Figs. 2 and 3). Using a mixed solvent system for the fluorescence measurements eliminated the difference between these samples. Dimethylformamide was chosen over other possible additives for both ease in sample handling and spectroscopic advantages. Detergents will foam and some have high background fluorescence; alcohols and ketones can evaporate, changing the concentration of the sample during measurements. The mixed solvent results in an enhanced fluorescence signal, increasing the sensitivity of the measurements.
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This allowed the use of shorter path lengths to avoid inner filter effects, extending the linear range over those reported previously (20, 28). The reproducibility of the assay is good, especially when standard curves are included for comparison. The error bars shown in Fig. 8 for melittin are representative of the deviations commonly observed for other peptide sequences and standards with or without lipid present. In common with other analytical assays, certain precautions will improve both accuracy and precision. Using Hamilton syringes, rather that air-displacement pipets, to measure and transfer samples for acid hydrolysis markedly decreases the deviations due to volumetric error. The various amino acid derivatives differ in their fluorescence intensities (22; Figs. 4 and 5) and standards with the same amino acid composition as the proteins or peptides being quantitated are advised for the greatest accuracy. Acid hydrolysis and neutralization should be carried out on standards as well as samples. The fluorescamine derivatives are stable in 40% DMF at room temperature; little to no change in intensity was seen over a period of a week. The fluorescence intensities do degrade with constant illumination at excitation wavelengths (Fig. 9) but exposure to room lighting for a week had no noticeable effect. The sensitivity of fluorescamine-based assays has
FIG. 8. Variability of assay results for the hydrolysis of melittin. Single samples of a 2 mg/ml stock melittin solution in deionized water (86% pure, Sigma Chemical Co.) were diluted to different concentrations and then hydrolyzed according to procedures outlined under Materials and Methods. Triplicate aliquots from the acid hydrolysis were then neutralized and derivatized with fluorescamine, and the fluorescence was measured in 40% DMF. Error bars indicate the standard deviation of these triplicate measurements. The linear regression on the intensity values (solid line) gives an R value of 0.9985.
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ACKNOWLEDGMENTS The author gratefully acknowledges Professor Thomas E. Thompson for his encouragement and support of this work and reading of the manuscript. This work was funded by NIH Grant GM-18628 to Professor T. E. Thompson.
REFERENCES
FIG. 9. Photodegradation of fluorescamine–alanine under constant illumination at 397 nm. Samples contained 20–30 nmol of fluorescamine–alanine. See Materials and Methods for details of sample preparation and spectroscopy.
been explored by other authors (20); this assay is based in part on the results of this previous work. As expected, the sensitivity of the assay is altered by substances that compete for fluorescamine reagent and increase the background fluorescence. However, the assay can reliably detect 2 nmol of amino acid in 4-ml assay volumes when the initial samples contain 0.1 M Mops or Hepes buffer and 0.3 M POPC. While other common substances containing reactive amino groups, such as nucleotides, nucleosides, and Tris buffer, were not tested, potential interference due to reagent consumption should be eliminated by using higher concentrations of fluorescamine stock solutions. Attempts to modify the assay for use with the amino-containing lipids phosphatidylethanolamine and phosphatidylserine were unsuccessful. The amino groups on both lipids can be blocked by acetylation or succinylation, but the blocking groups are unstable to acid hydrolysis. As discussed above, these lipids will interfere with the assay both by reducing the amount of fluorescamine available to react with amino acid and by contributing a large background fluorescence. It may be possible to correct for small amounts of these lipids by using high concentrations of fluorescamine solutions to form derivatives and estimating the lipid fluorescence contribution using separate samples containing only lipid, but this was not tested in this report.
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