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Artificial reductant enhancement of the Lowry method for protein determination
ANALYTICAL
BIOCHEMISTRY
155,243-248
( 1986)
Artificial Reductant Enhancement of the Lowry Method for Protein Determination ERIC LARSON, Plant Biology
Section,
BRUCE HOWLETT,
Plant Science
Building,
AND ANDRE
Cornell
University,
JAGENDORF Ithaca,
New York
14853
Received October 11, 1985 Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used. D 1986 Academic Press. Inc. KEY WORDS: protein determination; Lowry method.
The Lowry method for measuring protein (1) has been a standard assay in biochemical laboratories over the past 35 years. Numerous modifications have been proposed, permitting the measurement of membrane proteins (2), avoiding interfering materials by prior precipitation of the protein (3), or allowing more stable absorbances (4), etc. However, as far as we know all procedures incorporate a 30-min waiting period to permit full color development after adding the Folin (phenol) reagent. We now report a procedure to eliminate most of that waiting period, thereby speeding up the measuring process. The modification also increases the absorbance of a given amount of most proteins by about 35 to 60%, compared to the original method. MATERIALS
AND
METHODS
Gelatin was from Baker and Adamson; other proteins and poly-amino acids were from Sigma. Approximate molecular weights were 23,000 for polylysine; 37,000 for the I: 1 copolymer of lysine, alanine; 90,000 for the 1: 1 copolymer of lysine, tyrosine; and 40,000 for polyornithine. The concentrations of gelatin and of the poly-amino acids were determined gravimetrically; those of the remaining proteins by absorbance at 280 nm. The extinction coefficients used for solutions at 1.0 mg/ml 243
were 0.667 for BSA,’ 1.7 1 for cytochrome c, 1.81 for carbonic anhydrase, 1.6 for trypsin, 0.75 for ovalbumin, and 1.O for both cu-casein and insulin (5). The proteins were dissolved and desalted into a buffer containing 25 mM phosphate, 25 mM NaCl at pH 7.5, except for insulin where the pH was raised to 9.5 and trypsin which was dissolved in 10 mM HCl. The Lowry C reagent ( 1) was made by mixing 1 part of 0.5% (w/v) CuSO, - 5H20 with 50 parts of 2% Na2C03, 0.1 N NaOH, 0.16% Na tartrate. RESULTS
The Folin reagent consists of a phosphomolybdate-tungstate complex in weak sulfuric acid (6). Its yellow color shows that it is in the oxidized form, similar to the better-known phosphomolybdate complex formed when measuring inorganic phosphate. These complexes are destroyed rapidly in basic solution, as seen by disappearance of the yellow color, after adding the Folin reagent to the very basic Lowry C reagent. After mixing with protein, appearance of the blue color indicates formation of the reduced complex, which is alkali stable. However, complete development of the blue color, i.e., full reduction, is rather slow with the conventional Lowry procedure. ’ Abbreviations used: BSA, bovine serum albumin; DTT. dithiothreitol. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
244
LARSON.
HOWLETT.
Our innovation is to wait 3 min for full destruction of any free, oxidized (yellow) complex; then add DTT or ascorbate for rapid complete reduction of phospho-molybdotungstate bound to protein. The resulting blue color is still proportional to the protein present. In the recommended revised procedure from 5 to 100 pg of protein (in 100 ~1 volume) is added to 1.0 ml of the Lowry C reagent. One hundred microliters of Folin reagent previously diluted 1: 1 with water is added, followed immediately by vortexing. After 3 min, 100 ~1 of 20 mM DTT is added, and the tube vortexed again. The absorbance of the solution is measured at 740 nm. Figure 1 shows the absorbance of a standard 50 pg of BSA when using increasing concentrations of dithiothreitol, and ascorbate. The curves have two very distinct regions: an initial sharp rise, then a range where color development increases slowly with higher levels of reductant. It seems likely that the reductant performs two functions, with different concentration response.
011
’ 0
1 20
STOCK
1
4.0
1
REOUCTANT
’
60
’
I
80
’
CONCENTRATION
’
’ IO 0
I ”
I 200
ImMl
FIG. I. Response of the Lowry assay to increasing concentrations of DTT and ascorbate. The concentrations of reductant shown on the graph are those in the 100 ~1 of stock reagent added; the final concentration in the (1.25 ml final volume) assay was 0.08 times the values shown. Data points at 30 mM in this experiment (not shown) had absorbance readings 3 to 4% higher. In other experiments 20 mM was the optimum concentration. Optical density at 740 nm was read 10 min after adding the reductant.
AND
JAGENDORF
Although ascorbate gives color development equivalent to or slightly better than DTT at high protein levels, protein concentration curves, when using ascorbate, show an anomalous nonlinearity between 0 and 5 pg. Its use is therefore not recommended. A few other reductants tested (sodium sulfite, phenylenediamine) were not satisfactory. It is likely, however, that an exhaustive survey would show a variety of different reductants that could perform this function effectively. Saturation of color development occurred when using 20 to 35 mM DTT in different experiments. The marginally greater color development possible with higher levels of DTT (5 to 15% above that when using a 10 mM stock concentration, as in most of the experiments shown in this paper) has to be balanced against the greater expense of using more DTT. We think a satisfactory compromise lies in the use of 20 tnM DTT, and this concentration is therefore recommended. Once the reductant is added, the blue color (absorbance at 740 nm) forms almost immediately, and is relatively stable (Table 1). During the first 2 h. for instance, the loss of absorbance compared to the 5-min reading was 2% or less; i.e., less than experimental error of the readings. Even at 3 h, the color loss amounted to only 3 to 6%. A slow decay of potential color occurs during the interval between Folin reagent addition and DTT addition (Fig. 2). There is an initial high absorbance representing color development from Folin reagent not complexed with protein. This decreases rapidly as the uncomplexed reagent decomposes in the alkaline solution. Its decomposition is largely complete in 1 min, with current reagents. Following the initial decline there is a steady loss at about 1% per min of the final absorbance due to protein (Fig. 2). Similar curves for this interval were found with a variety of proteins other than BSA. Standard curves for BSA using the original Lowry method and the revised procedure are shown in Fig. 3. The shapes of the two curves are very similar, with no improvement in lin-
MORE
RAPID LOWRY
245
METHOD
TABLE 1 STABILITY
OF THE BLUE
COLOR
FORMED
WITH
20
50 pg OF BSA
Time after adding DTT
OD at 74 nm
Loss (90)
5 min 10 min lh 2h 3h 4h 5h
0.954 + 0.01 I4 0.964 rfr 0.032 0.945 f 0.016 0.933 f 0.016 0.901 + 0.021 0.8 14 + 0.028 0.776 + 0.018
1.0 2.2 5.6 14.7 18.7
USING
THE LOWRY
DTT
mM
METHOD
WITH
50
DTT
mM
OD at 740 nm 0.982 0.987 0.962 0.967 0.952 0.928 0.885
DTT
ADDED
Loss
f 0.022 zk 0.024 f 0.02 1 + 0.02 1 f 0.020 + 0.02 1 f 0.001
(90)
2.1 1.7 3.1 5.5 9.9
’ Standard deviation of three replicates for each point.
earity due to using DTT. However, at any specific concentration of the protein, the absorbance is 40 to 45% higher with the revised procedure. In view of the nonlinearity of the curves, this comparison is not entirely valid. A better 250,,
I
,
I
I
,
I
I
I
,
,
,
2.25
0 TIME
procedure is to find the amount of protein needed to generate a standard absorbance value, arbitrarily set at 0.30 here. In Fig. 3 this is given by 18.2 pg of bovine serum albumin when using the original procedure, but by 11.7 pg in the revised procedure with dithiothreitol. If we define “specific absorbance” as absorbance units per microgram of protein, the values for BSA are 16.5 for the original method and 25.6 for the revised procedure (neglecting
.&-A-a 0-A 2 4 6 6 IO BETWEEN FOLIN AND O T T (MINI
FIG. 2. Absorbance of reagent blank, and of 50 pg BSA, as a function of time between adding the Folin reagent and adding DTT. Both samples were read against water in the reference position of the spectrophotometer. The BSA values were plotted after subtracting reagent blank values manually.
BSA
(pgm)
FIG. 3. Absorbance as a function of BSA concentration, comparing the original (no DTT) and revised (+DTT) Lowry methods.
246
LARSON,
HOWLETT,
AND
the slight volume difference between the two methods). The ratio of these is 1.55 for the revised vs original methods, i.e., about a 55% improvement. Similar concentration curves were measured for six other proteins, and the ratios of specific absorbances determined (Table 2). Even though the specific absorbances varied considerably (trypsin having an unusually high specific absorbance; casein and especially gelatin unusually low values); in all casesthe new method gave from 32 to 59% improvement. The very high amount of color produced by trypsin, and very low level by gelatin, were noted in the original paper of Lowry et al. (1). A similar analysis derived from concentration curves of four soluble poly-amino acids showed increases in color development between 42 and 63% (Table 3). In this group the copolymer of lysine and tyrosine was most efficient, as expected. Polyornithine, polylysine, and the copolymer of lysine and alanine re-
quired four to six times as much material to give the same amount of Lowry color. DISCUSSION
The Folin reagent was used originally for measuring phenols (6), so its ability to produce color in the protein assay was ascribed to the tyrosines present (1). Formation of the blue color was known to be due to its reduction, probably by the tyrosine hydroxyl groups. However, in the original Lowry paper it was noted that (a) the first-formed color had a somewhat different absorption spectrum than the final one, and (b) full color development took about 30 min. This was interpreted as the rapid formation of an initial reduced complex, followed by rearrangement to the final complex. In view of our present findings we would like to concur that the assay does indeed proceed in two sequential steps. However, we would suggest that the initial quaternary com-
TABLE COMPARISON
OF PROTEIN
ABSORBANCES
USING
JAGENDORF
2
THE ORIGINAL
AND MODIFIED
LOWRY PROCEDURES
Mass needed for Specific absorbancea
Ratiob
11.7
16.5 25.6
1.55
16.3 10.5
18.0 28.6
I .59
29.5 19.5
10.2 16.4
1.51
47.1 31.5
6.4 9.5
1.48
16.4 11.8
18.3 25.4
1.39
18.2 13.6
16.5 22.1
1.34
8.7 6.6
34.5 45.5
1.32
,4,&l = 0.30
Protein
DTT
(rg)
Bovine serum albumin
+
18.2
Cytochrome c
-
Casein Gelatin Ovalbumin Insulin Trypsin
+
a Defined as A&mg protein unadjusted for volume differences. b Specific absorbance +DTT/-DTT.
247
MORE RAPID LOWRY METHOD TABLE 3 LOWRYCOLORREACTIONSOFSOMEPOLY-AMINO ACIDS Mass needed for Amino acid in polymer
A,g, = 0.30 DTT
Lysine Lysine, alanine Lysine, tyrosine
(PLg)
Specific absorbance”
Ratio*
+
54 33
5.6 9.1
1.63
+
41 25.5
1.3 11.8
1.61
31.5 52.2
1.39
8.1 11.5
I .42
+
Ornithine +
8.0 5.75 31 26.2
’ Defined as A,&mg protein. * Specific absorbance +DTT/-DTT.
plex between the polypeptide and the (P-MoW) Folin reagent is reasonably but not completely stable in alkali and either not reduced or only partially reduced. Evidence for partial instability lies in the slow decline in potential color yield during the time between adding the Folin reagent and adding dithiothreitol (once the initial rapid decomposition of uncomplexed Folin reagent is complete-see Fig. 2). We can further suggest that the second step is reduction of the primary complex to the fully reduced form, rather than a rearrangement. Evidence for this is the fact that DTT or ascorbate speeds up color development so that it is complete in seconds rather than requiring 30 min. Adding the reductant may increase the color yield because it eliminates most of the time during which slow decomposition of the initial complex can occur. As expected from its use as a reagent for measuring phenols, the copolymer of lysine and tyrosine gave four to six times the color yield (specific absorbance) as did polylysine, polyornithine, or poly(lysine, alanine). However, the fact that the latter three produced any color at all, especially without DTT, would not be expected if the reagent were completely specific for phenols. We can suggest that they
were able to form a weak complex with the Folin reagent, whose extent is best indicated from the result with DTT added. When DTT is not added, reduction of the Folin reagent must occur by means of electrons from the polypeptides decomposing under the highly alkaline conditions of the test. Oxidation during the Lowry assay of peptide bonds in polypeptides without reducing side chains was demonstrated directly in a paper (7) which appeared after this manuscript was submitted. The greater color yield from peptides containing aromatic amino acids could be due to either more efficient formation of the postulated initial complex, or greater efficiency in reducing the Folin reagent, or a combination of the two. However, complete reduction of the Folin reagent (without added DTT) was not more rapid for poly(lysine, tyrosine) than for the others. Also the percentage of improvement due to adding DTT was almost the same for polypeptides with and without tyrosine (Table 2). These facts make it seem likely to us that tyrosine especially favors rapid formation of a strong initial complex, in addition to being a better reductant than other amino acids. With the revised procedure, immediate, complete mixing of the Folin reagent with the
248
LARSON, HOWLETT,
Lowry C solution is more critical than before. The acidic Folin reagent is more dense than the basic Lowry C solution. If not mixed fully, some will fall to the bottom of the tube where both acidic pH and intact yellow phosphomolybdo-tungstate complex can be maintained. Added DTT will reduce the intact free complex, giving anomalous and very high absorbance values. The higher C&O4 level (4% stock solution) used by Markwell et al. (2) for membrane proteins is not recommended with the revised procedure. Excess copper interacts with both DTT and ascorbate leading to formation of a precipitate, resulting in lower apparent protein values. The higher level of copper also caused a slower breakdown of the free Folin complex; 3 min were then required for elimination of excess color in the blank solutions. We ordinarily use the method of coprecipitation of the protein with deoxycholate in trichloroacetic acid, introduced by Bensadoun and Weinstein (3), to avoid interfering chemicals. However, we found that a lo-min centrifugation rather than the recommended 30 min gave complete sedimentation of the protein and highest color yields. With these simple revisions the Lowry assay for protein is made considerably more rapid and convenient, and incidentally a little more sensitive. The relative color yield of different proteins is largely unaltered (Table 1) and we
AND JAGENDORF
believe this indicates that the fundamental basis for color development has not been changed. From our data, more emphasis has to be placed on stabilization of the Folin reagent by adsorption to the protein than on the number of reducing groups available in the protein (since these are now added in large excess in our revised procedure). The modification should be generally useful, and ought to make it simpler to adapt the test for automated methodologies. ACKNOWLEDGMENT This work was supported in part by Grant GM-14479 from the National Institutes of Health.
REFERENCES I. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. (195 I) J. Biol. Chem. 193,265-275. 2. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert. N. E. (1978) Anal. Biochem. 87,206-2 IO. 3. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70,24 I-250. 4. Ohnishi, S. T., and Barr, J. K. (1978) Anal. Biochem. 86. 193-200. 5. Sober. E. K. (1978) in CRC Handbook of Biochemistry: Selected Data for Molecular Biology (Sober, H. A., ed.), 2nd ed.. pp. C71-C99, Chem. Rubber Pub. Co., Cleveland, Ohio. 6. Folin, O., and Ciocalteau. V. (1927) J. Biol. Chem. 73,627-650.
7. Legler, G., Muller-Platz. C. M.. Mentges-Hettkamp, M.. Pflieger. G.. and Julich. E. (1985) Anal. Biochem. 150,278-287.
BIOCHEMISTRY
155,243-248
( 1986)
Artificial Reductant Enhancement of the Lowry Method for Protein Determination ERIC LARSON, Plant Biology
Section,
BRUCE HOWLETT,
Plant Science
Building,
AND ANDRE
Cornell
University,
JAGENDORF Ithaca,
New York
14853
Received October 11, 1985 Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used. D 1986 Academic Press. Inc. KEY WORDS: protein determination; Lowry method.
The Lowry method for measuring protein (1) has been a standard assay in biochemical laboratories over the past 35 years. Numerous modifications have been proposed, permitting the measurement of membrane proteins (2), avoiding interfering materials by prior precipitation of the protein (3), or allowing more stable absorbances (4), etc. However, as far as we know all procedures incorporate a 30-min waiting period to permit full color development after adding the Folin (phenol) reagent. We now report a procedure to eliminate most of that waiting period, thereby speeding up the measuring process. The modification also increases the absorbance of a given amount of most proteins by about 35 to 60%, compared to the original method. MATERIALS
AND
METHODS
Gelatin was from Baker and Adamson; other proteins and poly-amino acids were from Sigma. Approximate molecular weights were 23,000 for polylysine; 37,000 for the I: 1 copolymer of lysine, alanine; 90,000 for the 1: 1 copolymer of lysine, tyrosine; and 40,000 for polyornithine. The concentrations of gelatin and of the poly-amino acids were determined gravimetrically; those of the remaining proteins by absorbance at 280 nm. The extinction coefficients used for solutions at 1.0 mg/ml 243
were 0.667 for BSA,’ 1.7 1 for cytochrome c, 1.81 for carbonic anhydrase, 1.6 for trypsin, 0.75 for ovalbumin, and 1.O for both cu-casein and insulin (5). The proteins were dissolved and desalted into a buffer containing 25 mM phosphate, 25 mM NaCl at pH 7.5, except for insulin where the pH was raised to 9.5 and trypsin which was dissolved in 10 mM HCl. The Lowry C reagent ( 1) was made by mixing 1 part of 0.5% (w/v) CuSO, - 5H20 with 50 parts of 2% Na2C03, 0.1 N NaOH, 0.16% Na tartrate. RESULTS
The Folin reagent consists of a phosphomolybdate-tungstate complex in weak sulfuric acid (6). Its yellow color shows that it is in the oxidized form, similar to the better-known phosphomolybdate complex formed when measuring inorganic phosphate. These complexes are destroyed rapidly in basic solution, as seen by disappearance of the yellow color, after adding the Folin reagent to the very basic Lowry C reagent. After mixing with protein, appearance of the blue color indicates formation of the reduced complex, which is alkali stable. However, complete development of the blue color, i.e., full reduction, is rather slow with the conventional Lowry procedure. ’ Abbreviations used: BSA, bovine serum albumin; DTT. dithiothreitol. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
244
LARSON.
HOWLETT.
Our innovation is to wait 3 min for full destruction of any free, oxidized (yellow) complex; then add DTT or ascorbate for rapid complete reduction of phospho-molybdotungstate bound to protein. The resulting blue color is still proportional to the protein present. In the recommended revised procedure from 5 to 100 pg of protein (in 100 ~1 volume) is added to 1.0 ml of the Lowry C reagent. One hundred microliters of Folin reagent previously diluted 1: 1 with water is added, followed immediately by vortexing. After 3 min, 100 ~1 of 20 mM DTT is added, and the tube vortexed again. The absorbance of the solution is measured at 740 nm. Figure 1 shows the absorbance of a standard 50 pg of BSA when using increasing concentrations of dithiothreitol, and ascorbate. The curves have two very distinct regions: an initial sharp rise, then a range where color development increases slowly with higher levels of reductant. It seems likely that the reductant performs two functions, with different concentration response.
011
’ 0
1 20
STOCK
1
4.0
1
REOUCTANT
’
60
’
I
80
’
CONCENTRATION
’
’ IO 0
I ”
I 200
ImMl
FIG. I. Response of the Lowry assay to increasing concentrations of DTT and ascorbate. The concentrations of reductant shown on the graph are those in the 100 ~1 of stock reagent added; the final concentration in the (1.25 ml final volume) assay was 0.08 times the values shown. Data points at 30 mM in this experiment (not shown) had absorbance readings 3 to 4% higher. In other experiments 20 mM was the optimum concentration. Optical density at 740 nm was read 10 min after adding the reductant.
AND
JAGENDORF
Although ascorbate gives color development equivalent to or slightly better than DTT at high protein levels, protein concentration curves, when using ascorbate, show an anomalous nonlinearity between 0 and 5 pg. Its use is therefore not recommended. A few other reductants tested (sodium sulfite, phenylenediamine) were not satisfactory. It is likely, however, that an exhaustive survey would show a variety of different reductants that could perform this function effectively. Saturation of color development occurred when using 20 to 35 mM DTT in different experiments. The marginally greater color development possible with higher levels of DTT (5 to 15% above that when using a 10 mM stock concentration, as in most of the experiments shown in this paper) has to be balanced against the greater expense of using more DTT. We think a satisfactory compromise lies in the use of 20 tnM DTT, and this concentration is therefore recommended. Once the reductant is added, the blue color (absorbance at 740 nm) forms almost immediately, and is relatively stable (Table 1). During the first 2 h. for instance, the loss of absorbance compared to the 5-min reading was 2% or less; i.e., less than experimental error of the readings. Even at 3 h, the color loss amounted to only 3 to 6%. A slow decay of potential color occurs during the interval between Folin reagent addition and DTT addition (Fig. 2). There is an initial high absorbance representing color development from Folin reagent not complexed with protein. This decreases rapidly as the uncomplexed reagent decomposes in the alkaline solution. Its decomposition is largely complete in 1 min, with current reagents. Following the initial decline there is a steady loss at about 1% per min of the final absorbance due to protein (Fig. 2). Similar curves for this interval were found with a variety of proteins other than BSA. Standard curves for BSA using the original Lowry method and the revised procedure are shown in Fig. 3. The shapes of the two curves are very similar, with no improvement in lin-
MORE
RAPID LOWRY
245
METHOD
TABLE 1 STABILITY
OF THE BLUE
COLOR
FORMED
WITH
20
50 pg OF BSA
Time after adding DTT
OD at 74 nm
Loss (90)
5 min 10 min lh 2h 3h 4h 5h
0.954 + 0.01 I4 0.964 rfr 0.032 0.945 f 0.016 0.933 f 0.016 0.901 + 0.021 0.8 14 + 0.028 0.776 + 0.018
1.0 2.2 5.6 14.7 18.7
USING
THE LOWRY
DTT
mM
METHOD
WITH
50
DTT
mM
OD at 740 nm 0.982 0.987 0.962 0.967 0.952 0.928 0.885
DTT
ADDED
Loss
f 0.022 zk 0.024 f 0.02 1 + 0.02 1 f 0.020 + 0.02 1 f 0.001
(90)
2.1 1.7 3.1 5.5 9.9
’ Standard deviation of three replicates for each point.
earity due to using DTT. However, at any specific concentration of the protein, the absorbance is 40 to 45% higher with the revised procedure. In view of the nonlinearity of the curves, this comparison is not entirely valid. A better 250,,
I
,
I
I
,
I
I
I
,
,
,
2.25
0 TIME
procedure is to find the amount of protein needed to generate a standard absorbance value, arbitrarily set at 0.30 here. In Fig. 3 this is given by 18.2 pg of bovine serum albumin when using the original procedure, but by 11.7 pg in the revised procedure with dithiothreitol. If we define “specific absorbance” as absorbance units per microgram of protein, the values for BSA are 16.5 for the original method and 25.6 for the revised procedure (neglecting
.&-A-a 0-A 2 4 6 6 IO BETWEEN FOLIN AND O T T (MINI
FIG. 2. Absorbance of reagent blank, and of 50 pg BSA, as a function of time between adding the Folin reagent and adding DTT. Both samples were read against water in the reference position of the spectrophotometer. The BSA values were plotted after subtracting reagent blank values manually.
BSA
(pgm)
FIG. 3. Absorbance as a function of BSA concentration, comparing the original (no DTT) and revised (+DTT) Lowry methods.
246
LARSON,
HOWLETT,
AND
the slight volume difference between the two methods). The ratio of these is 1.55 for the revised vs original methods, i.e., about a 55% improvement. Similar concentration curves were measured for six other proteins, and the ratios of specific absorbances determined (Table 2). Even though the specific absorbances varied considerably (trypsin having an unusually high specific absorbance; casein and especially gelatin unusually low values); in all casesthe new method gave from 32 to 59% improvement. The very high amount of color produced by trypsin, and very low level by gelatin, were noted in the original paper of Lowry et al. (1). A similar analysis derived from concentration curves of four soluble poly-amino acids showed increases in color development between 42 and 63% (Table 3). In this group the copolymer of lysine and tyrosine was most efficient, as expected. Polyornithine, polylysine, and the copolymer of lysine and alanine re-
quired four to six times as much material to give the same amount of Lowry color. DISCUSSION
The Folin reagent was used originally for measuring phenols (6), so its ability to produce color in the protein assay was ascribed to the tyrosines present (1). Formation of the blue color was known to be due to its reduction, probably by the tyrosine hydroxyl groups. However, in the original Lowry paper it was noted that (a) the first-formed color had a somewhat different absorption spectrum than the final one, and (b) full color development took about 30 min. This was interpreted as the rapid formation of an initial reduced complex, followed by rearrangement to the final complex. In view of our present findings we would like to concur that the assay does indeed proceed in two sequential steps. However, we would suggest that the initial quaternary com-
TABLE COMPARISON
OF PROTEIN
ABSORBANCES
USING
JAGENDORF
2
THE ORIGINAL
AND MODIFIED
LOWRY PROCEDURES
Mass needed for Specific absorbancea
Ratiob
11.7
16.5 25.6
1.55
16.3 10.5
18.0 28.6
I .59
29.5 19.5
10.2 16.4
1.51
47.1 31.5
6.4 9.5
1.48
16.4 11.8
18.3 25.4
1.39
18.2 13.6
16.5 22.1
1.34
8.7 6.6
34.5 45.5
1.32
,4,&l = 0.30
Protein
DTT
(rg)
Bovine serum albumin
+
18.2
Cytochrome c
-
Casein Gelatin Ovalbumin Insulin Trypsin
+
a Defined as A&mg protein unadjusted for volume differences. b Specific absorbance +DTT/-DTT.
247
MORE RAPID LOWRY METHOD TABLE 3 LOWRYCOLORREACTIONSOFSOMEPOLY-AMINO ACIDS Mass needed for Amino acid in polymer
A,g, = 0.30 DTT
Lysine Lysine, alanine Lysine, tyrosine
(PLg)
Specific absorbance”
Ratio*
+
54 33
5.6 9.1
1.63
+
41 25.5
1.3 11.8
1.61
31.5 52.2
1.39
8.1 11.5
I .42
+
Ornithine +
8.0 5.75 31 26.2
’ Defined as A,&mg protein. * Specific absorbance +DTT/-DTT.
plex between the polypeptide and the (P-MoW) Folin reagent is reasonably but not completely stable in alkali and either not reduced or only partially reduced. Evidence for partial instability lies in the slow decline in potential color yield during the time between adding the Folin reagent and adding dithiothreitol (once the initial rapid decomposition of uncomplexed Folin reagent is complete-see Fig. 2). We can further suggest that the second step is reduction of the primary complex to the fully reduced form, rather than a rearrangement. Evidence for this is the fact that DTT or ascorbate speeds up color development so that it is complete in seconds rather than requiring 30 min. Adding the reductant may increase the color yield because it eliminates most of the time during which slow decomposition of the initial complex can occur. As expected from its use as a reagent for measuring phenols, the copolymer of lysine and tyrosine gave four to six times the color yield (specific absorbance) as did polylysine, polyornithine, or poly(lysine, alanine). However, the fact that the latter three produced any color at all, especially without DTT, would not be expected if the reagent were completely specific for phenols. We can suggest that they
were able to form a weak complex with the Folin reagent, whose extent is best indicated from the result with DTT added. When DTT is not added, reduction of the Folin reagent must occur by means of electrons from the polypeptides decomposing under the highly alkaline conditions of the test. Oxidation during the Lowry assay of peptide bonds in polypeptides without reducing side chains was demonstrated directly in a paper (7) which appeared after this manuscript was submitted. The greater color yield from peptides containing aromatic amino acids could be due to either more efficient formation of the postulated initial complex, or greater efficiency in reducing the Folin reagent, or a combination of the two. However, complete reduction of the Folin reagent (without added DTT) was not more rapid for poly(lysine, tyrosine) than for the others. Also the percentage of improvement due to adding DTT was almost the same for polypeptides with and without tyrosine (Table 2). These facts make it seem likely to us that tyrosine especially favors rapid formation of a strong initial complex, in addition to being a better reductant than other amino acids. With the revised procedure, immediate, complete mixing of the Folin reagent with the
248
LARSON, HOWLETT,
Lowry C solution is more critical than before. The acidic Folin reagent is more dense than the basic Lowry C solution. If not mixed fully, some will fall to the bottom of the tube where both acidic pH and intact yellow phosphomolybdo-tungstate complex can be maintained. Added DTT will reduce the intact free complex, giving anomalous and very high absorbance values. The higher C&O4 level (4% stock solution) used by Markwell et al. (2) for membrane proteins is not recommended with the revised procedure. Excess copper interacts with both DTT and ascorbate leading to formation of a precipitate, resulting in lower apparent protein values. The higher level of copper also caused a slower breakdown of the free Folin complex; 3 min were then required for elimination of excess color in the blank solutions. We ordinarily use the method of coprecipitation of the protein with deoxycholate in trichloroacetic acid, introduced by Bensadoun and Weinstein (3), to avoid interfering chemicals. However, we found that a lo-min centrifugation rather than the recommended 30 min gave complete sedimentation of the protein and highest color yields. With these simple revisions the Lowry assay for protein is made considerably more rapid and convenient, and incidentally a little more sensitive. The relative color yield of different proteins is largely unaltered (Table 1) and we
AND JAGENDORF
believe this indicates that the fundamental basis for color development has not been changed. From our data, more emphasis has to be placed on stabilization of the Folin reagent by adsorption to the protein than on the number of reducing groups available in the protein (since these are now added in large excess in our revised procedure). The modification should be generally useful, and ought to make it simpler to adapt the test for automated methodologies. ACKNOWLEDGMENT This work was supported in part by Grant GM-14479 from the National Institutes of Health.
REFERENCES I. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. (195 I) J. Biol. Chem. 193,265-275. 2. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert. N. E. (1978) Anal. Biochem. 87,206-2 IO. 3. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70,24 I-250. 4. Ohnishi, S. T., and Barr, J. K. (1978) Anal. Biochem. 86. 193-200. 5. Sober. E. K. (1978) in CRC Handbook of Biochemistry: Selected Data for Molecular Biology (Sober, H. A., ed.), 2nd ed.. pp. C71-C99, Chem. Rubber Pub. Co., Cleveland, Ohio. 6. Folin, O., and Ciocalteau. V. (1927) J. Biol. Chem. 73,627-650.
7. Legler, G., Muller-Platz. C. M.. Mentges-Hettkamp, M.. Pflieger. G.. and Julich. E. (1985) Anal. Biochem. 150,278-287.