of Avidin and Streptavidin HARMESHKSHARMA'
by a Modified
ANDCLAUDETIHON P.O. Box 2004, Mishawaka.
Received September 8, 1987 Underestimation of avidin and streptavidin by the Bradford method can be alleviated by carrying out the reaction at 100°C for 10-l 5 min, and the protein values thus obtained match very well with those of biotin-binding assays.“Thermal modification” is applicable to “standard” and “micro” versions of the method and has no effect on the spectral and stability characteristics of the protein-dye complexes. Q 1988 Academic press, Inc. KEY WORDS: avidin; streptavidin; thermal modification; Bradford method.
The recent popularity of the Bradford method (1) of calorimetric protein estimation can be attributed to its simplicity and the commercial availability of the reagent. In this method, the dye Coomassie brilliant blue G-250 binds to a protein in its “leuko” form (2) and the protein-dye complex is quantitated calorimetrically using a suitable protein standard. The method provides good results with proteins in which the number and accessibility of their “dye-binding sites” are similar to those of the standard protein. However, these conditions are not always met satisfactorily and there are several proteins which react poorly with the dye (3-7). During the standardization of the avidinbiotin complex system for immunohistochemical and immunocytochemical purposes, it was observed that avidin and strep tavidin react slowly and incompletely with the dye and that these problems could not be corrected by several modifications of the Bradford method (3,5-7). In this report, a “thermal modification,” which not only provides accurate estimates of the two proteins but also retains the simplicity and rapidity of the original method, is described.
Bovine serum albumin (BSA)2 was obtained from Miles Diagnostics (Kankakee, IL). Avidin, biotin, and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were from Sigma. Streptavidin was from Scripps Lab. The Bradford reagent was purchased from Bio-Rad Laboratories. All other reagents were of analytical grade. Proteins were dissolved in phosphate-buffered saline and centrifuged at 20,OOOg for 10 min, and their concentrations in supernatants were determined spectrophotometrically using A:& nm values as follows: BSA, 6.6 (8) avidin 15.4 (9), streptavidin 34 (10). Avidin and streptavidin values were confirmed by spectrophotometric titrations with biotin (11) and HABA (12). The three methods provided consistent results with *5% variation, and the average values were used as the exact concentrations of the stock solutions in this paper. “Standard” and “micro” versions of the Bradford method were carried out as described (1). For the “thermal modification,” the procedure was the same except that the ‘Abbreviations used: BSA, bovine serum albumin; TNBS, 2,4,6-trinitrobenzenesulfonic acid; HABA, 2-(4’hydroxyazobenzene)benzoic acid.
’ To whom all correspondence should be addressed. 135
Copyright Q 1988 by Academic Press, Inc. AU rigba of reproduction in any form rserved.
% Relative sensitivity Method
“Standard” and “micro” Bradfordb Modification of the Bradford method a. Macart and Gerbaut (5) b. Duhamel et al. (6) c. Sedmak and Grossberg (7) (i) HC104 (ii) HCl d. Read and Northcote (4) (i) 0.005% dye (ii) 0.01% dye Bromphenol blue 2,4,6-Trinitrobenzenesulfonate Lowry Modified Lowry Alkaline ninhydrin Microbiuret
85-95 60-70 67-80 113-119 146-162 88-122 100-105 95-98
80-90 60-75 70-80 116-124 200-219 85-125 100-105 95-102
a Relative sensitivity is the percentage ratio of the amount of avidin or streptavidin determined by a particular method and the amount determined by the methods described under Materials and Methods. The data are from two different experiments, each run in triplicate. b The values were obtained after incubating the reaction mixture for 60 min.
reaction mixture containing appropriate amounts of avidinlstreptavidin and the dye were kept for lo- 15 min in a boiling-water bath and then cooled to room temperature and its A595 “,,, was monitored within lo15 min. Other calorimetric procedures used were Lowry et al. ( 13), modified Lowry ( 14), microbiuret (15), alkaline ninhydrin (16), bromphenol blue ( 17), and TNBS ( 18). BSA was used as a standard in the normal and the thermally modified Bradford assays. RESULTS AND DISCUSSION
Based upon the high affinity of biotin ( lo-” M) for avidin and streptavidin, several methods have been developed for their quantitation ( 1 1- 12,19 and reference therein). Though precise, these methods are time-consuming and sophisticated and do not match the convenience of calorimetric
methods of protein determination. On the other hand, as shown in Table 1, colorimetric methods either under- or overestimate avidin and streptavidin. For example, the Bradford method gives 30-40% less protein values; the extent of underestimation was even greater when the protein solutions were stored at 4°C for a month or longer. Several modifications of the method, such as the addition of minute amounts of sodium dodecyl sulfate (6,7) or carrying out the reaction in different acids (7) as well as at different dye concentrations (4), do not provide accurate estimates of the proteins. Similar conclusions can be drawn when the proteins are measured by the bromphenol blue, the TNBS, the Lowry, and the modified Lowry methods (Table 1). The microbiuret and the ninhydrin methods give accurate results, but they require large protein concentrations. As mentioned above, neither avidin nor streptavidin maintained a consistent dye re-
sponse upon storage, thus making both unsuitable as standards. In contrast, this was not the case with BSA, and its use as a standard was therefore considered more appropriate. Table 2 shows that the reaction between the Bradford dye and the avidin/streptavidin improves as the temperature is increased, reaching a maximum at 100°C within lo- 15 min. Further heating precipitates the protein-dye complexes, thereby causing a 10-20s decrease at A595 nm. Neither BSA nor the reagent control exhibited an equivalent increase at -4595nm. Figure 1 shows the effect of “thermal treatment” on different concentrations of avidin and streptavidin in the “standard” and the “micro” Bradford method. The reaction is linear up to 80 /Ig in the standard (Fig. IA) and up to 20 pg in the micro method (Fig. 1B). The data were further analyzed with respect to the amount of protein added and the amount obtained with and without the thermal treatment. There was a linear relationship between the two, and the slopes of the lines in the standard method were 1.O + 0.05 with thermal treatment and 0.54
OF TEMPERATURE ON THE DYE REACHON AA 595 nm
Conditions of the Bradford reaction
15 min at 23’C 60°C 80°C
at 100°C for 5 min 10 min 15 min 30 min
0.27 1 0.283 0.316
0.258 0.264 0.279
0.332 0.390 0.364 0.332
0.323 0.383 0.389 0.332
Note. Avidin (28 &assay) and streptavidin (27 pg,/ assay) were used. The values are the averages of experiments run in triplicate.
f 0.1 without thermal treatment. The values for the micro method were 1.06 f 0.08 with thermal treatment and 0.70 + 0.1 without thermal treatment. When the AsY5,,,,, of the reaction mixture after thermal treatment was continuously monitored, it was observed that it did not change for approximately 60 min and decreased by only 10-l 5% in the following 60 min. These results indicate that the thermally treated samples should not be stored beyond 60 min. The thermal treatment changes the typical Bradford color to malachite green, and the spectrophotometric measurements shown in Fig. 2 reveal that at 80°C the “orange” form (X,,, 467 nm) is converted to the “blue” form of the dye (X,;, 650 nm) (Curves 1 and 2) and the reaction can be reversed by lowering the temperature (Curve 3). Similarly, the spectrum of the avidin-dye complex was dominated by the blue form of the dye at 80°C and when the temperature was lowered the spectrum became qualitatively identical to that of the unheated sample (Curves 4-6). The only difference was that the thermally treated sample possessed more absorption in the range of 400-750 nm, and this observation was further reinforced with the difference spectra of the avidin-dye complex against the dye (curves 7 and 8). It should be noted that for avidin as well as for streptavidin (data not shown) the X,,, of 595 nm remained unaffected by the 80°C thermal treatment. These results show that the color developed after the thermal treatment can be safely monitored at 595 nm. It is possible that increased amounts of the blue form of the dye produced by thermal treatment have a role to play in the increased dye response of avidin and streptavidin. This was tested by determining avidin and streptavidin concentrations with solutions in which different amounts of the blue species were produced by varying the amounts of phosphoric acid and ethanol (4). It was observed that solutions containing more blue
E 8 :
FIG. 1. Increasing concentrations of avidin (0) and streptavidin (A) were measured in the “standard” (A) and the “micro” assay (B). Results with and without the “thermal treatment” are given by the closed and open symbols, respectively.
dye species did not improve the dye response with both the proteins. The other alternative, that more dye-binding sites are exposed by the thermal treat-
ment, was evaluated in an experiment in which 40 pg of avidin and streptavidin was heated in a boiling-water bath for 10 min and then cooled to room temperature, and their
1.5 P a B 9
FIG. 2. Visible spectra of the dye against water at room temperature (Curve I), at 8O’C (Curve 2), and after cooling to room temperature (Curve 3). Spectra of 50 fig avidin plus the dye against water at room temperature (Curve 4), at 80°C (Curve 5), and after cooling to room temperature (Curve 6). Difference spectra of 50 rg avidin plus the dye against the dye at room temperature (Curve 7) and after cooling to room temperature following a 80°C “thermal treatment” (Curve 8).
protein contents were determined by the conventional Bradford method. It was observed that the A595 nm increased from 0.15 to 0.434 for streptavidin and decreased from 0.38 to 0.2 for avidin, thus indicating that more dye-binding sites of streptavidin became available upon heating, which is in contrast to avidin where they had been denatured. An explanation for why the avidin gives an increased dye response during the thermal treatment may be that the avidindye complex is more heat stable than the avidin alone. Attempts to expose the “buried dye-binding sites” of both proteins by treatments such as incubation with increasing concentrations of urea, guanidinium chloride, 0.2% sodium taurocholate, 0.1 N NaOH, or HCl were not successful. Furthermore, these treatments were not effective after the reduction of proteins with 0.1 M 2-mercaptoethanol. Thermal modification as described in this report may find application with proteins where dye-binding sites are inaccessible under normal Bradford conditions. The treatment is reminiscent of a modification of the Lowry method (20) in which incubation of proteins with copper-tar&ate complex at 100°C for 100 min improved the color intensity. ACKNOWLEDGMENT The authors acknowledge Ms. Debra Ebling for skillfully typing the manuscript.
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