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Lowry method of protein quantification: Evidence for photosensitivity
ANALYTICAL
BRXHEMISTRY
14,
391-393 (1984)
Lowry Method of Protein Quantification:
Evidence
JANET M. DAWSONANDPAUL
for Photosensitivity
L.HEATLIE
Department of Biology, Oxford Polytechnic, Headington. Oxford Received August 1, 1983 Despite reports of its susceptibility to various interfering factors, the Fohn Phenol protein quantification method of 0. H. Lowry, N. J. Rosebrough, A. L. Fan; and R. J. RandaU (1951, J. Biol. Chem. 193, 265-275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken. KEY WORDS: Lowry assay; protein assay;photosensitivity; light; temperature; protein.
The most widely used procedure for the quantitative determination of protein is that of Lowry, Rosenbrough, Fat-r, and Randall (1). Since this method was developed, several interfering substances have been identified (for review, see (2)). Various modifications have been devised to overcome interference by such substances, while maintaining the simplicity, sensitivity, and precision for which this assay is noted. While using the method of Lowry et al. (1) and the modification described by Markwell et al. (3) for routine determination of protein concentration, it became apparent that some factor other than protein concentration was affecting the final absorbance readings. As absorbance values were consistently higher in samples which were directly exposed to natural light, it was reasoned that the assay may be photosensitive. The present study demonstrates the photosensitive nature of the Lowry protein assay and suggests precautions which should be taken to overcome the problem. MATERIALS
AND METHODS
All reagents were obtained from Sigma Limited, London, and the same reagents were used throughout the investigation.
Protein was measured by the method of Lowry et al. (1) using bovine serum albumin (BSA).’ Standard curves were prepared in the concentration range 0- 100 @g/ml BSA. The assay was performed in glass test tubes (16 X 150 mm) arranged in open-sided test tube racks, partitioned into a front and rear compartment by an opaque plastic septum. Replicate tubes were placed in the two compartments allowing exposure to different natural light intensities during the two assay incubation stages ( 15 and 30 min, respectively). Initial experiments were conducted on the bench, at room temperature. The assay was also performed in a glasswalled waterbath maintained at 27 -t 1°C. Under these conditions the assay was performed both in bright sunlight and in a reduced natural light condition by shielding the water bath from direct sunlight. Light intensity and temperature were monitored in the same position throughout the investigation using a Type E22 megatron light meter (Megatron Limited, England) and mercury bulb thermometers. Absorbances were read at 600 nm on a Cecil UV spectrophotometer. Statistical analyses were performed using ’ Abbreviation used: BSA, bovine serum albumin.
391
OOO3-2697/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.
392
DAWSON
the statistical package SFANOVA mented on a Prime 750 computer.
AND HEATLIE
imple-
RESULTS AND DISCUSSION
When the Lowry procedure was performed on the bench at room temperature, statistically significant differences were observed between replicate protein samples, the differences being dependent upon their relative position in the test tube rack (front exposed and rear shielded). It is felt that the methodology followed is typical of many other laboratories which routinely use this assay. The differences were reasoned to be a consequence of photosensitivity. It was, however, possible that the differences might be accounted for in terms of differential temperature effects. Miller (4) has shown that increasing temperature (5O”C,lO min) accelerates the development of the final chromogen. The possibility of temperature differences between the tube positions accounting for the changes in absorbance was eliminated by conducting all further experiments under controlled temperature conditions. In order to investigate the effect of light intensity on the final development of the chromogen, a range of protein concentrations was exposed to high and to low natural light intensities (35,700 f 4600 and 52.4 f 11.0 lux). The absorbance values were verified statistically in a 3-way analysis of variance. The factors examined were protein concentration, light (light or reduced light), and position (front or back). A significant main effect of light F( 1,20) = 90.19, p < 0.001 in addition to the expected main effect of concentration F(4,20) = 836.34, p < 0.001 was obtained. Light was also significant as an interaction with concentration F( 1,20) = 5.32, p < 0.001, reflecting larger differences between the light and reduced light conditions at higher concentrations. Why this should be is unclear. Position was not significant as a main effect(F = 0.01, & = 1,20) but was significant as an interaction with light E’( 1,20) = 8.4, p < 0.00 1.
This effect may be accounted for as a result of the procedure used to effect the reduced light condition. In the light condition, the front (exposed) tubes exhibited higher absorbance values but in the reduced light condition, the rear tubes were higher. This reversal is believed to be due to the light shielding in the water bath which cast the front of the rack into deeper shade than the back. Thus, the shielded compartment in the light condition became relatively light exposed in the reduced light condition. Performing the assay in complete darkness supports this assumption to the extent that no effect of position was observed. These findings have significant implications for routine protein determinations where large numbers of samples are assayed simultaneously. On the bench, up to 10% discrepancy in estimated protein concentration occurred between replicate protein samples positioned in the front and back of the test tube rack. The possibility of such discrepancy is especially important in absolute amounts, when considering that protein samples frequently require dilutions of up to lOOO-fold to attain a protein concentration in the linear range of the Lowry assay (20-100 &ml). The absence of a position effect when the assay was performed at constant temperature in a water bath was possibly due to the light having to travel through additional media as compared to the bench where the position effect was significant (F(l,lO) = 15.67, p -c 0.01). Although it appears there are conditions under which the assay is photosensitive, further characterization of the effect is still required. The stage at which the assay is photosensitive, and the wavelength and intensity of the light required to produce an effect remain uncertain. The latter is of great practical importance as it is not altogether clear whether the assay is affected by artificial lighting or if the effect is restricted to natural sunlight at an undetermined intensity. Until further research suggests the most appropriate methodology, the authors do have a preferred procedure which does not involve working in the
LOWRY
ASSAY PHOTOSENSITIVITY
dark. This simply involves ensuring that all samples, including the standards, are subjected to the same level of illumination at the time the assay is performed. It is felt that this procedure should ensure the assay maintains both its convenience and accuracy. ACKNOWLEDGMENTS We gratefully acknowledge Stephen Fearnley for the use of SFANOVA and statistical advice, and Dr. C. J. B. White and Dr. D. A. Fell for their valued comments.
393
Both authors are supported by grants from the Science and Engineering Research Council.
REFERENCES 1. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275.
Peterson, G. L. ( 1979) Anal. Biochem. 100,20 l-220. 3. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. ( 1978) Anal. Biochem. 87,206-2 10. 4. Miller, G. L. (1959) Anal. Chem. 31, 964. 2.
BRXHEMISTRY
14,
391-393 (1984)
Lowry Method of Protein Quantification:
Evidence
JANET M. DAWSONANDPAUL
for Photosensitivity
L.HEATLIE
Department of Biology, Oxford Polytechnic, Headington. Oxford Received August 1, 1983 Despite reports of its susceptibility to various interfering factors, the Fohn Phenol protein quantification method of 0. H. Lowry, N. J. Rosebrough, A. L. Fan; and R. J. RandaU (1951, J. Biol. Chem. 193, 265-275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken. KEY WORDS: Lowry assay; protein assay;photosensitivity; light; temperature; protein.
The most widely used procedure for the quantitative determination of protein is that of Lowry, Rosenbrough, Fat-r, and Randall (1). Since this method was developed, several interfering substances have been identified (for review, see (2)). Various modifications have been devised to overcome interference by such substances, while maintaining the simplicity, sensitivity, and precision for which this assay is noted. While using the method of Lowry et al. (1) and the modification described by Markwell et al. (3) for routine determination of protein concentration, it became apparent that some factor other than protein concentration was affecting the final absorbance readings. As absorbance values were consistently higher in samples which were directly exposed to natural light, it was reasoned that the assay may be photosensitive. The present study demonstrates the photosensitive nature of the Lowry protein assay and suggests precautions which should be taken to overcome the problem. MATERIALS
AND METHODS
All reagents were obtained from Sigma Limited, London, and the same reagents were used throughout the investigation.
Protein was measured by the method of Lowry et al. (1) using bovine serum albumin (BSA).’ Standard curves were prepared in the concentration range 0- 100 @g/ml BSA. The assay was performed in glass test tubes (16 X 150 mm) arranged in open-sided test tube racks, partitioned into a front and rear compartment by an opaque plastic septum. Replicate tubes were placed in the two compartments allowing exposure to different natural light intensities during the two assay incubation stages ( 15 and 30 min, respectively). Initial experiments were conducted on the bench, at room temperature. The assay was also performed in a glasswalled waterbath maintained at 27 -t 1°C. Under these conditions the assay was performed both in bright sunlight and in a reduced natural light condition by shielding the water bath from direct sunlight. Light intensity and temperature were monitored in the same position throughout the investigation using a Type E22 megatron light meter (Megatron Limited, England) and mercury bulb thermometers. Absorbances were read at 600 nm on a Cecil UV spectrophotometer. Statistical analyses were performed using ’ Abbreviation used: BSA, bovine serum albumin.
391
OOO3-2697/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.
392
DAWSON
the statistical package SFANOVA mented on a Prime 750 computer.
AND HEATLIE
imple-
RESULTS AND DISCUSSION
When the Lowry procedure was performed on the bench at room temperature, statistically significant differences were observed between replicate protein samples, the differences being dependent upon their relative position in the test tube rack (front exposed and rear shielded). It is felt that the methodology followed is typical of many other laboratories which routinely use this assay. The differences were reasoned to be a consequence of photosensitivity. It was, however, possible that the differences might be accounted for in terms of differential temperature effects. Miller (4) has shown that increasing temperature (5O”C,lO min) accelerates the development of the final chromogen. The possibility of temperature differences between the tube positions accounting for the changes in absorbance was eliminated by conducting all further experiments under controlled temperature conditions. In order to investigate the effect of light intensity on the final development of the chromogen, a range of protein concentrations was exposed to high and to low natural light intensities (35,700 f 4600 and 52.4 f 11.0 lux). The absorbance values were verified statistically in a 3-way analysis of variance. The factors examined were protein concentration, light (light or reduced light), and position (front or back). A significant main effect of light F( 1,20) = 90.19, p < 0.001 in addition to the expected main effect of concentration F(4,20) = 836.34, p < 0.001 was obtained. Light was also significant as an interaction with concentration F( 1,20) = 5.32, p < 0.001, reflecting larger differences between the light and reduced light conditions at higher concentrations. Why this should be is unclear. Position was not significant as a main effect(F = 0.01, & = 1,20) but was significant as an interaction with light E’( 1,20) = 8.4, p < 0.00 1.
This effect may be accounted for as a result of the procedure used to effect the reduced light condition. In the light condition, the front (exposed) tubes exhibited higher absorbance values but in the reduced light condition, the rear tubes were higher. This reversal is believed to be due to the light shielding in the water bath which cast the front of the rack into deeper shade than the back. Thus, the shielded compartment in the light condition became relatively light exposed in the reduced light condition. Performing the assay in complete darkness supports this assumption to the extent that no effect of position was observed. These findings have significant implications for routine protein determinations where large numbers of samples are assayed simultaneously. On the bench, up to 10% discrepancy in estimated protein concentration occurred between replicate protein samples positioned in the front and back of the test tube rack. The possibility of such discrepancy is especially important in absolute amounts, when considering that protein samples frequently require dilutions of up to lOOO-fold to attain a protein concentration in the linear range of the Lowry assay (20-100 &ml). The absence of a position effect when the assay was performed at constant temperature in a water bath was possibly due to the light having to travel through additional media as compared to the bench where the position effect was significant (F(l,lO) = 15.67, p -c 0.01). Although it appears there are conditions under which the assay is photosensitive, further characterization of the effect is still required. The stage at which the assay is photosensitive, and the wavelength and intensity of the light required to produce an effect remain uncertain. The latter is of great practical importance as it is not altogether clear whether the assay is affected by artificial lighting or if the effect is restricted to natural sunlight at an undetermined intensity. Until further research suggests the most appropriate methodology, the authors do have a preferred procedure which does not involve working in the
LOWRY
ASSAY PHOTOSENSITIVITY
dark. This simply involves ensuring that all samples, including the standards, are subjected to the same level of illumination at the time the assay is performed. It is felt that this procedure should ensure the assay maintains both its convenience and accuracy. ACKNOWLEDGMENTS We gratefully acknowledge Stephen Fearnley for the use of SFANOVA and statistical advice, and Dr. C. J. B. White and Dr. D. A. Fell for their valued comments.
393
Both authors are supported by grants from the Science and Engineering Research Council.
REFERENCES 1. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275.
Peterson, G. L. ( 1979) Anal. Biochem. 100,20 l-220. 3. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. ( 1978) Anal. Biochem. 87,206-2 10. 4. Miller, G. L. (1959) Anal. Chem. 31, 964. 2.