- Email: [email protected]
Measurement of protein concentration with interferences optics
ANALYTICAL
BIOCHEMISTRY
28, 216-221
Measurement
of
(1969)
Protein
Interferences
JORGE BABUL Department
of
Biochemistry,
AND
Concentration
with
Optics’
EARLE
University
Received July
STELLWAGEN
of Iowa,
Iowa
City, Iowa
52260
10, 1968
Measurement of the physical and chemical properties of a purified protein requires a method for determination of the protein concentrations of the solutions employed. Spectrophotometric measurements are widely used as a convenient, sensitive, and precise method for the determination of protein concentration. However, spectrophotometric measurements require a knowledge of an extinction coefficient for each protein, since the relative number of chromophoric amino acid residues varies from protein to protein. Determination of an extinction coefficient requires an independent and precise method of measuring protein concentration. The limited supply of many purified enzymes precludes the use of dry weight measurements in determining the extinction coefficient. Although chemical methods for protein determination, such as the Kjeldahl, Folin-Ciocalteu, or biuret procedures, require much less protein, such measurements must be related to a standard reference protein. Since each of these chemical methods is sensitive to the relative abundance of specific amino acid residues in proteins (l-3)) an arbitrary standard protein such as bovine serum albumin may be inappropriate for a given protein of unknown composition. Although the molar refractions of the amino acid residues also exhibit considerable variation (4)) the refractive indices of proteins vary by less than 62% (4,5). Accordingly, refractometric measurements can be used to determine protein concentration. This communication describes the use of an analytical ultracentrifuge as a differential refractometer to determine the concentration of small volumes of dilute protein solutions. 1 This investigation was supported by research grant GB-6288 from the National Science Foundation and by a Public Health Service research program award l-K03-GM 08’737 from the Institute of General Medical Sciences. 216
MEASUREMENT MATERIALS
OF
PROTEIN AND
CONCENTRATION
217
METHODS
Proteins. Ribonuclease A, trypsin, chymotrypsin, and lactate dehydrogenase were purchased from Worthington Biochemical Corp., serum albumin and cytochrome c from Sigma Chemical Co., and aldolase from Boehringer und Soehne. Ovalbumin and methemoglobin were gifts from Dr. H. B. Bull. All proteins except trypsin were dialyzed against 0.05 M phosphate buffer, pH 7.0. Trypsin was dialyzed against 0.05 M tris-HCl buffer, pH 8.0. All solutions were clarified by centrifugation to remove any insoluble material. Ultracentrifugal menswrements. A 0.15 ml aliquot of a protein solution was placed in one limb of a 12 mm double-sector synthetic boundary centerpiece assembled in a cell together with sapphire windows, and 0.42 ml of the outer dialysis solution in the other limb of the centerpiece. All centrifugation was performed in a Spinco model E analytical ultracentrifuge at 20’ using a Wratten No. 77A filter, Solvent was layered over the solution by accelerating a rotor containing the cell to a speed of less than 10,000 rpm to form the solvent-protein boundary. The rotor was then maintained at this speed to permit the boundary to diffuse. When individual fringes could be discerned across the boundary in the viewing screen, a photograph of the cell contents was taken using Kodak spectroscopic II-G photographic plates. The rotor was then stopped, uncoupled from the centrifuge, inverted several times to destroy any protein gradients, reaccelerated to the operating speed, and photographed to provide a baseline exposure to correct for any optical distortion of the interference fringes. The number of interference fringes traversed when crossing the protein-solvent boundary was measured with the aid of a Nikon microcomparator. Spect~ophotometric measurements. The absorbance of aliquots of all solutions was measured at 280 rnp except those of cytochrome c and methemoglobin, which were measured at 550 and 500 rnp, respectively. A Gilford spectrophotometer was used. Protein concentrations were calculated using the extinction coefficients shown in Table 1. RESULTS
AND
DISCUSSION
The number of interference fringes observed for each protein exhibited a linear dependence on protein concentration in the range 0.1 to 5.0 mg/ml. A typical example is shown in Figure 1. Protein concentrations less than 0.1 mg/ml produced interference patterns exhibiting curvature throughout the cell, suggesting that the small difference in the densities of the solution and solvent at these low
218
BABUL
Refractive Protein
Refractive increment, measured (fringes/mg/ml)
Aldolase, rabbit muscle a-Chymotrypsin, bovine pancreas Lactate dehydrogenase, bovine heart Ovalbumin, chicken Ribonuclease A, bovine pancreas Serum albumin, bovine Trypsin, bovine pancreas Cytochrome c, horse heart Methemoglobin, bovine
AND
STELLWAGEN
TABLE 1 Increments of Proteinsa Properties
----
Ref&d;y ealcula&d
Partial volume
ax, I%-‘, cm-1
specific (ml/gmf
Amino acid composition
4.06 4.04
1.593 1.596
9.38 18.7
( 6)
0.742 0.736
( 7) ( 9)
( 8) (10)
4.14
1.604
14.9
(11)
0.740
(12)
(13 1
4.03 4.23
1.594 1.613
7.146 6.95 (15)
0.745 0.709
(14) (14)
(10) (10)
4.15 4.06 4.43 4.50
1.604 1.595 1.584c 1.586”
6.60 (16) 14.4 (17) 22.6 (20) 5.32”
0.734 0.730 0.724 0.749
(14) (18)
(10) (19) (10) 00)
(14)
a Numbers in parentheses are references for the values employed in the calculations. b H. B. Bull, personal communication. c Calculated for apoprotein.
protein concentrations cause convective stirring during the layering of solvent. The refractive increment in fringes/mg/ml calculated from the slope of the observed linear relationship for each protein is shown in Table 1. The seven nonheme proteins have an average refractive increment of 4.10 f 0.13 interference fringes/mg/ml, while the two hemoproteins have an average value of 4.47 I+ 0.04 fringes/mg/ml. The refractive index of each of the seven nonheme proteins was calculated from their amino acid compositions, partial specific volumes, and refraction per gram residue (4) using the Lorentz and Lorenz equation as described by McMeekin et al. (4). As shown in Figure 2, the observed refractive increment exhibits a linear dependence on the calculated refractive indices, indicating that the variation in refractive increment is due to the differences in amino acid composition and not to procedural errors. Assuming that the seven nonheme proteins examined represent a normal variation of amino acid composition, measurement of the concentration of an unknown unconjugated protein should not be in error by more than -3% using an ultracentrifuge as a differential refractometer and the reported refractive increment. This procedure can be employed to determine the concentration of protein solutions containing as little as 0.02 mg of protein in a volume of 0.15 ml.
MEASUREMENT
Protein
OF PROTEIN
Concentration
CONCENTRATION
219
, mg /ml
FIG. 1. Dependence of number of interference fringes on protein concentration. A solution of lactic dehydrogenase was dialyzed against 0.05 1M phosphate buffer, pH 7.0. Dilutions of the dialyzed protein solution were made with the outer dialysis solution. Protein concentrations were determined spectrophotometrically using the extinction coefficient shown in Table 1.
Prosthetic groups would be expected to contribute to the refractive increment in accordance with their molar refraction and fractional concentration. As shown in Table 1 and Figure 2, the refractive increments of the hemoproteins, cytochrome c and methemoglobin, are substantially greater than predicted from their amino acid compositions. Since these proteins contain only 4-60/o heme by weight, the refraction per gram of heme must be markedly different from that of an average amino acid residue. Therefore, to utilize this method for the measurement of the concentration of conjugated proteins, it will be necessary to evaluate the fractional weight concentration of the conjugated moiety and its refraction per gram. SUMMARY
A convenient micro method for the measurement centration that uses an analytical ultracentrifuge
of protein conas a differential
220
BABUL
AND
STELLWAGEN
“62r~
REFRACTIVE
2. Comparison of refractive conjugated proteins. The refractive composition as described in Table the data. FIG.
INCREMENT
(fringes/
mg/ ml)
increment and refractive index for the unindex was calculated from the amino acid 1. The line shown is a least-squares fit of
refractometer is described. The number of interference fringes observed for a given protein solution is converted to mg protein/ml using an average refractive increment of 4.1 fringes/mg/ml. REFERENCES 1. KIRK, P. L., Advan. Protein Chem. 3, 139 (1947). A. G., BARDAWILL, C. J., AND DAVD, M. M., J. Biol. Chem. 177, 2. GORNALL, 751 (1959). R. F., AND GILL, D. M., Anal. Bioclzem. 9, 401 (1964). 3. ITZHAKI, 4. MCMEEKIN, T. L., WILENSKY, M., AND GROVES, M. L., Biochem. Biophys. Res. Commun. 7, 151 (1962). E. P., in “The Proteins” (H. Neurath and K. 5. DOTY, P., AND GEIDUSCHEK, Bailey, eds.), Vol. I, Part A, p. 393. Academic Press, New York, 1953. 6. DONOVAN, J. W., Biochemistry 3, 67 (1964). J. F., AND LOWRY, C., Biochim. Biophys. Acta 20, 109 (1956). 7. TAYLOR, (P. D. Boyer, H. Lardy, and K. Myr8. RUTTER, W. J., in “The Enzymes” btick, eds.), Vol. 5, 2nd ed., p. 341. Academic Press, New York, 1961. 9.
SCHWERT,
G. W.,
AND
KAUFMAN,
S., J.
Biol.
Chem.
190,
807
(1951).
G. R., AND SMITH, R. H., Advan. Protein Chem. 18, 227 (1963). 10. TRISTRAM, R. H., STOLZENBACH, F., CAHN, R. D., AND KAPLAN, 11. PESCE, A., MCKAY, N. O., J. Biol. Chem. 239, 1753 (1964). E., AND MARKERT, C. L., Biochem. Biophys. Res. Commun. 6, 12. APPELLA, 171
(1961). A., FONDY, J. Biol. CILem.
13. PESCE,
T.,
STOLZENBACH,
242, 2151 (1967).
F.,
CASTILLO,
F. AND
KAPLAN,
N.
O.,
MEASUREMENT 14. 15. 16. 17. 18. 19. 20.
OF
PROTEIN
CONCENTRATION
221
EDSALL, J. T., in “The Proteins” (H. Neurath and K. Bailey, eds.) , Vol I, Part B. p. 549. Academic Press, New York, 1953. SHERWOOD, L. M., AND POTTS, J. T., J. Biol. Chem. 240, 3799 (1965). DANIEL, E., AND WEBER, G., Biochemistry 5, 1893 (1966). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 50’7 (1955). CUNNINGHAIV~, L. W., JR., TIETZE, F., GREEN, N. M., AND NEURATH, H., Discuss. Faraday Sot. 13, 58 (1953). WALSH, K. A., AND NEURATH, H., PTOC. Natl. Acad. Sci. U. S. 52, 884 (1964). MARGOLIASH, E., AND FROHWIRT, N., Biochem. J. 71, 570 (1959).
BIOCHEMISTRY
28, 216-221
Measurement
of
(1969)
Protein
Interferences
JORGE BABUL Department
of
Biochemistry,
AND
Concentration
with
Optics’
EARLE
University
Received July
STELLWAGEN
of Iowa,
Iowa
City, Iowa
52260
10, 1968
Measurement of the physical and chemical properties of a purified protein requires a method for determination of the protein concentrations of the solutions employed. Spectrophotometric measurements are widely used as a convenient, sensitive, and precise method for the determination of protein concentration. However, spectrophotometric measurements require a knowledge of an extinction coefficient for each protein, since the relative number of chromophoric amino acid residues varies from protein to protein. Determination of an extinction coefficient requires an independent and precise method of measuring protein concentration. The limited supply of many purified enzymes precludes the use of dry weight measurements in determining the extinction coefficient. Although chemical methods for protein determination, such as the Kjeldahl, Folin-Ciocalteu, or biuret procedures, require much less protein, such measurements must be related to a standard reference protein. Since each of these chemical methods is sensitive to the relative abundance of specific amino acid residues in proteins (l-3)) an arbitrary standard protein such as bovine serum albumin may be inappropriate for a given protein of unknown composition. Although the molar refractions of the amino acid residues also exhibit considerable variation (4)) the refractive indices of proteins vary by less than 62% (4,5). Accordingly, refractometric measurements can be used to determine protein concentration. This communication describes the use of an analytical ultracentrifuge as a differential refractometer to determine the concentration of small volumes of dilute protein solutions. 1 This investigation was supported by research grant GB-6288 from the National Science Foundation and by a Public Health Service research program award l-K03-GM 08’737 from the Institute of General Medical Sciences. 216
MEASUREMENT MATERIALS
OF
PROTEIN AND
CONCENTRATION
217
METHODS
Proteins. Ribonuclease A, trypsin, chymotrypsin, and lactate dehydrogenase were purchased from Worthington Biochemical Corp., serum albumin and cytochrome c from Sigma Chemical Co., and aldolase from Boehringer und Soehne. Ovalbumin and methemoglobin were gifts from Dr. H. B. Bull. All proteins except trypsin were dialyzed against 0.05 M phosphate buffer, pH 7.0. Trypsin was dialyzed against 0.05 M tris-HCl buffer, pH 8.0. All solutions were clarified by centrifugation to remove any insoluble material. Ultracentrifugal menswrements. A 0.15 ml aliquot of a protein solution was placed in one limb of a 12 mm double-sector synthetic boundary centerpiece assembled in a cell together with sapphire windows, and 0.42 ml of the outer dialysis solution in the other limb of the centerpiece. All centrifugation was performed in a Spinco model E analytical ultracentrifuge at 20’ using a Wratten No. 77A filter, Solvent was layered over the solution by accelerating a rotor containing the cell to a speed of less than 10,000 rpm to form the solvent-protein boundary. The rotor was then maintained at this speed to permit the boundary to diffuse. When individual fringes could be discerned across the boundary in the viewing screen, a photograph of the cell contents was taken using Kodak spectroscopic II-G photographic plates. The rotor was then stopped, uncoupled from the centrifuge, inverted several times to destroy any protein gradients, reaccelerated to the operating speed, and photographed to provide a baseline exposure to correct for any optical distortion of the interference fringes. The number of interference fringes traversed when crossing the protein-solvent boundary was measured with the aid of a Nikon microcomparator. Spect~ophotometric measurements. The absorbance of aliquots of all solutions was measured at 280 rnp except those of cytochrome c and methemoglobin, which were measured at 550 and 500 rnp, respectively. A Gilford spectrophotometer was used. Protein concentrations were calculated using the extinction coefficients shown in Table 1. RESULTS
AND
DISCUSSION
The number of interference fringes observed for each protein exhibited a linear dependence on protein concentration in the range 0.1 to 5.0 mg/ml. A typical example is shown in Figure 1. Protein concentrations less than 0.1 mg/ml produced interference patterns exhibiting curvature throughout the cell, suggesting that the small difference in the densities of the solution and solvent at these low
218
BABUL
Refractive Protein
Refractive increment, measured (fringes/mg/ml)
Aldolase, rabbit muscle a-Chymotrypsin, bovine pancreas Lactate dehydrogenase, bovine heart Ovalbumin, chicken Ribonuclease A, bovine pancreas Serum albumin, bovine Trypsin, bovine pancreas Cytochrome c, horse heart Methemoglobin, bovine
AND
STELLWAGEN
TABLE 1 Increments of Proteinsa Properties
----
Ref&d;y ealcula&d
Partial volume
ax, I%-‘, cm-1
specific (ml/gmf
Amino acid composition
4.06 4.04
1.593 1.596
9.38 18.7
( 6)
0.742 0.736
( 7) ( 9)
( 8) (10)
4.14
1.604
14.9
(11)
0.740
(12)
(13 1
4.03 4.23
1.594 1.613
7.146 6.95 (15)
0.745 0.709
(14) (14)
(10) (10)
4.15 4.06 4.43 4.50
1.604 1.595 1.584c 1.586”
6.60 (16) 14.4 (17) 22.6 (20) 5.32”
0.734 0.730 0.724 0.749
(14) (18)
(10) (19) (10) 00)
(14)
a Numbers in parentheses are references for the values employed in the calculations. b H. B. Bull, personal communication. c Calculated for apoprotein.
protein concentrations cause convective stirring during the layering of solvent. The refractive increment in fringes/mg/ml calculated from the slope of the observed linear relationship for each protein is shown in Table 1. The seven nonheme proteins have an average refractive increment of 4.10 f 0.13 interference fringes/mg/ml, while the two hemoproteins have an average value of 4.47 I+ 0.04 fringes/mg/ml. The refractive index of each of the seven nonheme proteins was calculated from their amino acid compositions, partial specific volumes, and refraction per gram residue (4) using the Lorentz and Lorenz equation as described by McMeekin et al. (4). As shown in Figure 2, the observed refractive increment exhibits a linear dependence on the calculated refractive indices, indicating that the variation in refractive increment is due to the differences in amino acid composition and not to procedural errors. Assuming that the seven nonheme proteins examined represent a normal variation of amino acid composition, measurement of the concentration of an unknown unconjugated protein should not be in error by more than -3% using an ultracentrifuge as a differential refractometer and the reported refractive increment. This procedure can be employed to determine the concentration of protein solutions containing as little as 0.02 mg of protein in a volume of 0.15 ml.
MEASUREMENT
Protein
OF PROTEIN
Concentration
CONCENTRATION
219
, mg /ml
FIG. 1. Dependence of number of interference fringes on protein concentration. A solution of lactic dehydrogenase was dialyzed against 0.05 1M phosphate buffer, pH 7.0. Dilutions of the dialyzed protein solution were made with the outer dialysis solution. Protein concentrations were determined spectrophotometrically using the extinction coefficient shown in Table 1.
Prosthetic groups would be expected to contribute to the refractive increment in accordance with their molar refraction and fractional concentration. As shown in Table 1 and Figure 2, the refractive increments of the hemoproteins, cytochrome c and methemoglobin, are substantially greater than predicted from their amino acid compositions. Since these proteins contain only 4-60/o heme by weight, the refraction per gram of heme must be markedly different from that of an average amino acid residue. Therefore, to utilize this method for the measurement of the concentration of conjugated proteins, it will be necessary to evaluate the fractional weight concentration of the conjugated moiety and its refraction per gram. SUMMARY
A convenient micro method for the measurement centration that uses an analytical ultracentrifuge
of protein conas a differential
220
BABUL
AND
STELLWAGEN
“62r~
REFRACTIVE
2. Comparison of refractive conjugated proteins. The refractive composition as described in Table the data. FIG.
INCREMENT
(fringes/
mg/ ml)
increment and refractive index for the unindex was calculated from the amino acid 1. The line shown is a least-squares fit of
refractometer is described. The number of interference fringes observed for a given protein solution is converted to mg protein/ml using an average refractive increment of 4.1 fringes/mg/ml. REFERENCES 1. KIRK, P. L., Advan. Protein Chem. 3, 139 (1947). A. G., BARDAWILL, C. J., AND DAVD, M. M., J. Biol. Chem. 177, 2. GORNALL, 751 (1959). R. F., AND GILL, D. M., Anal. Bioclzem. 9, 401 (1964). 3. ITZHAKI, 4. MCMEEKIN, T. L., WILENSKY, M., AND GROVES, M. L., Biochem. Biophys. Res. Commun. 7, 151 (1962). E. P., in “The Proteins” (H. Neurath and K. 5. DOTY, P., AND GEIDUSCHEK, Bailey, eds.), Vol. I, Part A, p. 393. Academic Press, New York, 1953. 6. DONOVAN, J. W., Biochemistry 3, 67 (1964). J. F., AND LOWRY, C., Biochim. Biophys. Acta 20, 109 (1956). 7. TAYLOR, (P. D. Boyer, H. Lardy, and K. Myr8. RUTTER, W. J., in “The Enzymes” btick, eds.), Vol. 5, 2nd ed., p. 341. Academic Press, New York, 1961. 9.
SCHWERT,
G. W.,
AND
KAUFMAN,
S., J.
Biol.
Chem.
190,
807
(1951).
G. R., AND SMITH, R. H., Advan. Protein Chem. 18, 227 (1963). 10. TRISTRAM, R. H., STOLZENBACH, F., CAHN, R. D., AND KAPLAN, 11. PESCE, A., MCKAY, N. O., J. Biol. Chem. 239, 1753 (1964). E., AND MARKERT, C. L., Biochem. Biophys. Res. Commun. 6, 12. APPELLA, 171
(1961). A., FONDY, J. Biol. CILem.
13. PESCE,
T.,
STOLZENBACH,
242, 2151 (1967).
F.,
CASTILLO,
F. AND
KAPLAN,
N.
O.,
MEASUREMENT 14. 15. 16. 17. 18. 19. 20.
OF
PROTEIN
CONCENTRATION
221
EDSALL, J. T., in “The Proteins” (H. Neurath and K. Bailey, eds.) , Vol I, Part B. p. 549. Academic Press, New York, 1953. SHERWOOD, L. M., AND POTTS, J. T., J. Biol. Chem. 240, 3799 (1965). DANIEL, E., AND WEBER, G., Biochemistry 5, 1893 (1966). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 50’7 (1955). CUNNINGHAIV~, L. W., JR., TIETZE, F., GREEN, N. M., AND NEURATH, H., Discuss. Faraday Sot. 13, 58 (1953). WALSH, K. A., AND NEURATH, H., PTOC. Natl. Acad. Sci. U. S. 52, 884 (1964). MARGOLIASH, E., AND FROHWIRT, N., Biochem. J. 71, 570 (1959).