ANALYTICAL
BIOCHEMISTRY
96, 130-
138 (1979)
A Precise Method for the Quantitation of Proteins Taking Account Their Amino Acid Composition1
into
HANS-J• ACHIM HORSTMANN lnstitut
ftir
Physiologische D-8520
Chemie, Erlangen,
Universittit Erlangen-Niirnberg, Federal Republic of Germany
Fahrstrasse
17#
Received September 27, 1978 A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-4 solutions containing 1 to 100 pg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of. F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per ahquot of hydrolysate analyzed can be determined.
Various methods have been used for the determination of proteins in biological materials. The well-known calorimetric method developed by Hsien Wu in 1922 (1) and elaborated by Lowry and co-workers in 1951 (2) using the phenol reagent of Folin and Ciocalteu (3) is by far the most frequently used. There are other methods for protein measurement such as estimation of Kjeldahl nitrogen, the determination of peptide groups using the biuret reaction, or the determination of protein-bound dye. By using these methods, however, correct results can only be obtained if the amino acid composition of the protein taken as the reference for calibrating the method corresponds well with that composition of the protein to be determined. Since the 1 Presented in part at the Joint Autumn Meeting of the Biochemical Societies of the German Federal Republic, Switzerland and France, Freiburg, Germany, October Z-5, 1977, and an abstract has appeared in Hoppe-Seyler’s 2. Physiol. Chem. (1977) 358,
i222.
0003-2697/79/090130-09$02.00/O Copyright 10 1979 by Academic FWss, k, All rights of reproduction in any form reserved.
molecular weights of the amino acid residues composing a protein vary from 57 (glytine) to 186 (tryptophan), the true protein content of a biological sample can only be determined by a quantitative analysis of the amino acids released after total hydrolysis taking into consideration their different contributions to the total weight. As a result, I here present a method based on the ninhydrin reaction of hydrolysates of proteins. Since, with the exception of proline and hydroxyproline, the response at 570 nm of all amino acids occurring in proteins to the ninhydrin reagent is virtually the same (4),* the total concentration of amino acids in a hydrolysate can be determined without fractionation of the mixture. The protein mass in the sample is then calculated by multiplying the nanomoles of total amino acids found by a conversion factor corresponding to the z This is also valid for cystine, found in hydrotysates instead of cysteine. 130
PRECISE
QUANTITATION
average molecular weight of 1 nmol of the mixture of the amino acid residues released from the protein. This conversion factor has to be determined. In routine analyses of the same biological material, e.g., erythrocyte membranes, this can be done by quantitative amino acid analysis of the hydrolysate following standard procedures. In the case of pure proteins the conversion factor can be calculated from the amino acid composition reported in the literature. For the majority of proteins the factor lies between 0.11 and 0.12. MATERIALS
AND METHODS
Reagents
All chemicals were of analytical grade. They were purchased from E. Merck, Darmstadt: 9.5 M HCl, suprapure; NaOH; sodium acetate, anhydrous; Tic&, approx 15% solution; sodium citrate, tryst 2HZO; ethylene glycol monomethyl ether, and phenol. The standard amino acid calibration mixture for protein hydrolysates was from Bio-Rad Laboratories, Mtinchen (stock solution: 2.500 ? 0.004 pmol per milliliter of each component; cystine is considered as half-cysteine). Water was deionized. Proteins
Bovine serum albumin, chromatographically pure, was from Boehringer-Mannheim. Protamine sulfate, pure, was obtained from Serva, Heidelberg. Erythrocyte membranes were prepared from freshly drawn human blood (5). Trabecular tissue from human eyes was a gift from Dr. K. Sames, Anatomisches Institut, Erlangen. The tissue was repeatedly extracted with 1 M NaCl at pH 3 in order to remove soluble materials. Solutions
Sodium citrate buffer, 0.2 M, pH 3.2, containing 0.1% phenol. Ninhydrin reagent:
OF PROTEIN
131
Dissolve 25 g of ninhydrin in a solution containing 750 ml of ethylene glycol monomethyl ether, 375 ml of 4 M sodium acetate buffer (pH 5.51), 7.5 g of sodium citrate, and 375 ml of water. Filter the solution into the reagent flask of the amino acid analyzer and add 7.5 ml of a solution of about 15% Tic&. During these procedures the solution is gassed with nitrogen. After standing for at least 6 h the reagent is ready for use. Procedures Step 1: Hydrolysis of proteins. Fill 5 to 20 ~1 of sample solution or suspension containing 1 to 100 pg protein into fine capillary tubes as used for the determination of blood sugar (internal diameter 0.8 to 1.2 mm, not heparinized; Braun , Melsungen) . In order to determine the transferred volumes exactly, measure the length of the liquid column and the internal diameter of the capillary with utmost accuracy, using a measuring microscope (E. Leitz, Wetzlar), or weigh the filled capillary on a microbalance. Fill 1.4 times this volume of 9.5 M HCl into the capillary tube and fuse both its ends. Mix the contents by shaking. Hydrolyse for 24 h at 105’C with occasional mixing. Force the solution into one end of the capillary tube and cut the glass just above the meniscus. Transfer the hydrolysate into a small plastic vessel using a long-tipped capillary pipet operated by mouth. Rinse both ends of the capillary repeatedly with a total volume of 200 ~1 of water. In order to hydrolyze dry materials weigh from 0.3 to 2 mg of the sample into small glass vessels (7.5 x 70 mm), add 200 ~1 of 5.5 M HCI, gas with nitrogen, fuse the vessel, and heat in an oven for 24 h at 105C. Step 2: Removal of ammonia by d$fusion.
Add about 1.5 times the volume of 9.5 M HCl, as used in step 1, of 10 M NaOH to the hydrolysate and mix it. The final pH
132
HANS-JOACHIM
should be about 10. Place the vessel in an almost flat position into a desiccator containing 1 M sulfuric acid and evacuate until a pressure of 40 mm Hg is reached. The diffusion of ammonia is allowed to proceed at room temperature for at least 5 h. Then acidify the hydrolysate with a calculated volume of 9.5 M HCl and desiccate the contents to dryness in a desiccator containing PZOSand pellets of KOH. Step 3: Determination
of ninhydrin
color.
Dissolve the dry residue from step 2 in an appropriate volume (e.g., 50 to 500 ~1) of 0.2 M sodium citrate buffer, pH 3.2. The convenient final concentration of total amino acids is about 1 nmol per microliter, corresponding to about 0.1 pg of protein per microliter. The total amino acids are quantitated without fractionation using the continuousflow system of an amino acid analyzer (e.g., BioCal 200). In order to achieve this, the apparatus is quickly modified by mounting the sample injection block between the outlet of the resin column and the ninhydrin mixing chamber. The flow of the buffer is adjusted to 35 ml/h, and the flow of the ninhydrin reagent to 25 ml/h. The temperature of the resin column is 65’C. By this means the pressure waves caused by the buffer pump are smoothed down to zero before they arrive at the injection block. Inject 2 to 20 ~1 of the solution to be tested containing up to 15 nmol of amino acids into the running buffer stream via the sample injection block using a calibrated microsyringe. Injecting should be finished within 10 s and can be repeated every 5 min. Calibrate the ninhydrin reaction by injecting aliquots of 2 to 10 ~1 of the standard amino acid calibration mixture, containing 0.85 nmol of total amino acids in 1.0 ~1.~ z A mixture of equal amounts of the 17 amino acids found in acid hydrolysates simulates a hydrolysate of a protein better than a solution of one pure amino acid, e.g., leucine, since the slightly varying color yields obtained from the different amino acids are balanced out.
HORSTMANN TABLE
1
WEIGHT OF 1 nmol OF RESIDUE OF AMINO ACID FOUND IN HYDROLYSATES OF PROTEINS
Amino acid residue
i
1 2 3 4
Asp Thr Ser
b
bG3) 0.115 0.101 0.087
GIU
0.129
5
ROa
6 7 8
Ala
0.097 0.057 0.071
9 10 11 12 13 14 15 16 17 I8 19 20
GUY CYS
Val Met Be Leu Vr Phe LYS His A@ TV Asn Gin
0.103 0.099 0.131 0.113 0.113 0.163 0.147 0.128 0.138 0.154 0.186 0.114 0.128
a Hydroxyproline (b = 0.113) should be treated like proline by calculating F.
The absorbance is measured at 570 nm using a microcuvette with 14-mm light path. Prior to use all the ammonia from the calibration mixture has to be removed as given in step 2. Determine, e.g., by integration, the areas under the peaks resulting from the ninhydrin reaction of the unfractionated mixture of amino acids. The peak areas obtained from the calibration mixture are used to establish a standard curve by plotting the areas versus the nanomoles of amino acids injected. Preferably, least-mean-squares regression is calculated from these values using a programmable desk-top calculator. With the parameters for a straight-line relationship thus determined, the nanomoles of amino acids of the sample can then be computed directly from the observed areas. Step 4: Calculation
of the protein
mass.
The weight of a protein is the sum of the
PRECISE QUANTITATION
FIG. I. Diagram of analysis of total amino acids. Aliquots injected repeatedly. For further conditions see text.
weights of the amino acid residues the protein is composed of. If the amino acid composition is known, the mass of the protein can be calculated by multiplying the number of the nanomoles of total amino acids found by the average molecular weight (in pg) of the residues of that amino acid mixture. This average residue weight is called the weight equivalent (WE) and can be calculated as follows: WE = z (Ut.bi)y i=l
[II
where u is the mole fraction of amino acid i per mole of total amino acid composition and b the weight (in pg) of 1 nmol of this respective amino acid residue. Index i represents the at most 20 different amino acids normally occurring in proteins. Their b values are given in Table 1. However, during hydrolysis of a protein performed in HCl tryptophan is destroyed,
133
OF PROTEIN
of samples indicated
have been
cysteine is replaced by cystine, and asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively. Moreover, proline cannot be determined at 570 nm with the ninhydrin reaction applied in the method described. For this reason, a conversion factor F, derived from WE, must be used for the calculation of the protein mass in the analyzed sample. F is characteristic of a protein or a defined protein mixture (e.g., serum or cell organelles) and has to be determined once for all the following quantitations of the same material by quantitative amino acid analysis of an aliquot of the material according to the formula
PI
where c are the nanomoles
of specific
134
HANS-JOACHIM
amino acid i found in the analyzed aliquot and b is the weight (in pg) of 1 nmol of amino acid residue i (see Table l), If solutions of pure proteins of known amino acid composition (including tryptophan) are to be determined, F can be approximated to the value found experimentally after a hydrolysis time of 24 h according to the formula
where u is the mole fraction of the specific amino acid i per mole of total amino acid composition; for i and b see Table I. This relationship was validated for bovine serum albumin. Its F value, 0.1205, calculated from the known amino acid composition (6) according to formula [3], corresponds closely to the F value found experimentally, 0.121, using formula [2]. The protein content of the sample, analyzed according to steps 1 to 3, can now be calculated by multiplying F by the nanomoles of total amino acids found. RESULTS
Reproducibility
and Sensitivity
The peaks obtained by injection of identical aliquots of amino acid solutions are highly reproducible (Fig. 1). Plotting the areas of the peaks of the calibration mixture shown in the figure versus their corresponding amounts of total amino acids resulted in a linear regression line (y = 0.076~ - 0.027; r = 0.999). Using this equation the amino acid concentration of the hydrolysate of the human erythrocyte membranes injected was calculated from the peak areas obtained (X values) to 5.33 ? 0.06 nmol/ 10 ~1 (mean * SD). The coefficient of variation was 1.1% (n = 6). Figure 2 illustrates a standard curve obtained from 20 injections of different volumes of the calibration mixture. The response is linear up to 17 nmol of the amino
HORSTMANN
acids tested. The means and standard deviations for 12 calibration runs made on different days were: correlation coefficient = 0.996 ? 0.005; intercept = 0.004 k 0.023; slope = 0.073 2 0.004. In a typical assay, a sample of a hydrolysate of unknown amino acid concentration is escorted by at least two different volumes of the calibration mixture and the aliquots are injected five times each. The method measures amino acid concentrations as low as 0.3 nmol/lO ~1, corresponding to a protein concentration of approximately 0.03 pg/lO ~1 hydrolysate injected. Variability
of Conversion Factor F
As evidenced by Fig. 3 the conversion factor F varies considerably according to the amino acid composition of a protein. For most proteins F lies between 0.100 and 0.125. Extreme values are related to elastin (0.083) and to the protamines (e.g., salmin 0.147), respectively. The assay has been tested with two pure proteins and two defined biological materials selected for their divergent amino acid composition. Compared to the weight equivalent WE calculated according to formula [1] from the amino acid composition found (see Table 2), F is always of a higher value conditioned by the different proline content of the proteins investigated. Reliability
of the Method
The only reliable method for the determination of the amount of protein in a biological material is to sum up the weights of the amino acid residues found in the protein by quantitative amino acid analysis. Bearing in mind this basic condition I tested the reliability of the new method by dividing the hydrolysates of the selected proteins shown in Table 2 into two parts, A and B. Part A was analyzed by fractionation using an automatic amino acid analyzer and the amount of protein present in the
PRECISE QUANTITATION
AMINO
ACID
OF PROTEIN
135
(ramok)
FIG. 2. Standard curve obtained with the assay, using an amino acid calib~tion mixture of known concentration. The area under a peak is calculated by multiplying the maximum AAs7@nm of a peak by its width (mm) at half-maximum absorbance. The peak areas obtained are plotted versus the nanomoles of total amino acids applied. Each point results from three to four injections of the same amino acid amount. All values are within the circle diameters. The linear regression line is inserted.
sample was calculated by summing up the weights of all the amino acid residues found. F was calculated according to formula [Z] . Part B was treated as described under Materials and Methods, steps 2 to 3, The amount of protein was calculated by multiplying the nanomoles of total amino acids found at 570 nm in the u~ractionated mixture by the conversion factor F derived from the respective part A. Figure 4 illustrates the results of the comparison of the two methods of protein determination. Taking the protein amounts found in the respective parts A by quantitative amino
acid analysis as the actual values, a linear regression line for all B analyses (n = 86) can be calculated: y = 0.97% + 0.032; r = 0.982. The overall recovery of protein, irrespective of its amino acid composition, determined by the new method was 1.017 ztz0.046. Comparison
with Other Methods
The protein of different erythrocyte membrane preparations was also assayed by two other procedures, (i) the Lowry method, as modified by Wang and Smith (8) and, (ii) the Biuret method according to Futterman
136
HANS-JOACHIM
HORSTMANN
TABLE AMINO
ACID COMPOSITION, WEIGHT EQUIVALENT WE, AND CONVERSION PROTEINS OR PROTEIN MIXTURES TO TEST THE PROTEIN ASSAY
Amino acid ASP Thr Ser GhI Pro GlY Ala CYS Val Met Be Leu Tyr Phe LYS His kz HYP WP Q4 F @td
2
Trabecular tissuea
Erythrocyte membranea
7.0 3.8 5.8 11.1 8.7 20.5 9.4
9.2 5.4 7.2 13.8 5.6 6.7 8.1
5.4
6.9 1.7 4.7 12.8 2.1 4.4 4.8 2.3 4.4
3.3 6.7 2.2 2.9 4.3 1.1 4.6 3.0 0.100 0.113
Bovine serum aIbumin”
0.113 0.121
a Moles per 100 mol of amino acid composition. b WE and F were calculated from the amino acid composition formulas 1 and 2, respectively.
DISCUSSION
The method presented is especially suited for insoluble biological materials such as cell
Protaminea
9.9 6.1 4.8 14.2 6.3 2.8 8.2 3.8 5.8 0.4 2.0 10.2 3.0 4.5 11.4 2.8 3.7
0.110 0.116
and Rollins (9), each time using bovine serum albumin as the standard. Referred to the protein content of these samples calculated from total amino acid analysis, only about 0.8 was found with the Lowry method and only about 0.5 with the Biuret method, respectively (IO). These findings show that using serum albumin as the reference protein makes it impossible to quantitate the protein of the erythrocyte membrane by these two methods. Notice that the weight equivalents of serum albumin and of the membrane protein mixture are very similar and, also, that their amino acid compositions are not strikingly different (see Table 2).
FACTOR F, FOR FOUR DESCRIBED
found in the hydrolysates
6.9 12.0 5.0 1.0 3.6 0.8
70.6 0.134 0.153 according to
organelles and for tissue probes available in small amounts. For the precise determination of the protein content of these samples only 5 to IO ~1 of suspension or solution containing as little as 100 ng of protein are needed. This procedure has been successfully applied by us to comparative studies of the peptide patterns obtained by enzymatic digestion of isolated membrane proteins. Using this technique it is necessary to know exactly both the ratio of protease to protein and the amounts of the peptides loaded onto polyacrylamide gradient gels.4 During hydrolysis of proteins asparagine and glutamine are split into ammonia and aspartic acid and glutamic acid, respectively. By this, however, the weights of these residues remain practically unchanged d Anselstetter, V. (1979) J. Chromafogr.,
172, 49-56.
PRECISE QUANTITATION
137
OF PROTEIN
Free amino acids, oligopeptides, amino sugars, and other soluble substances yielding color at 570 nm with ninhydrin interfere with this protein determination. For this reason, these substances must be removed from the sample prior to hydrolysis. Cell organelles and tissue probes should be washed repeatedly by suspension in a suitable buffer and collected by centrifugation. Soluble proteins can be separated from interfering low-molecular-weight material by trichloroacetic acid precipitation, using the method of Bensadoun and Weinstein (11) in the case of very dilute samples. Besides ninhydrin other reagents for the determination of a-amino groups may be used provided that all amino acids reacting with the reagent give the same color response. This may be the case for fluorescamine (Hoffmann-La Roche). In this way the
r
e 0.06
k 0.10
ha]
1 c 0 12
P
014
FIG. 3. Distribution of the conversion factor F of 93 different proteins. F is the weight (in pg) of 1 nmol of the mixture of amino acid residues composing a specific protein. The amino acid composition for most of the proteins selected were taken from the data compilated by Kirschenbaum [e.g., ref. (7)] using the formula [3] for the calculation of E. The positions of some proteins are indicated: a = bovine serum albumin; c = casein: e = elastin (bovine aorta); h = hemoglobin; k = collagen; p = protamine.
(see Table 1). Cysteic acid obtained by oxidation of cysteine residues should be taken as cysteine when calculating F. An amino acid analyzer slightly modified as described is used for the assay, but should not be a prerequisite. Any spectrophotometer adapted to measure a small volume (e.g., 0.1 ml) in a cuvette with a long light path should be suitable as well. However, for the determination of the conversion factor F an amino acid analyzer is needed unless F is otherwise known.
PROTEIN (pg),
method
A
FIG. 4. Comparison of two different methods for the determination of protein. Method A: protein found by quantitative amino acid analysis; method B: protein found by the assay described. The linear regression line is inserted. For further information see text. (0) Trabecular tissue, human eye; (0) human erythrocyte membrane; (A) bovine serum albumin; (A) protamine. Each symbol represents the average of two to six injections of the same ahquot of hydrolysate. Vertical lines indicate the range when these values are larger than the symbol diameters.
138
HANS-JOACHIM
sensitivity of the assay may be increased considerably. ACKNOWLEDGMENTS I wish to express my gratitude to Dr. Volker Anselstetter for helpful discussions and critical commerits. This work was supported by a grant (Ho 118/l 1) from the Deutsche Forschungsgemeinschaft.
REFERENCES 1. Wu, H. (1922)J. Bioi. Chem. Sl, 33-39. 2. Lowry, 0. H., Rosebrough, H. I., Farr, A. L., and Randall, R. I. (1951) J. BioL Chem. 193, 265-275. 3. Folin, O., and Ciocalteu, V. (1927) J. Bio/. Chem. 73, 627-650.
HORSTMANN
4. Moore, (Them.
S., and Stein, W. H. (1954) J. Biol. 211,907-913.
5. Anselstetter, V., and Horstmann, H. J. (1975) Eur. J. Biochem. S6, 259-269. 6. Spahr, P. F., and Edsall, J. T. (1964) J. Biol. Chem. 239, 850-854. 7. Kirschenbaum, D. M. (1977) Anal. Biochem. 83, 521-550. 8. Wang, C. S., and Smith, R. L. (1975) Anal. Biochem. 63, 414-417. 9. Futterman, S., and Rollins, M. H. (1973) Ana/. Biochem. Sl, 443-447. 10. Stierstorfer, S., Horstmann, H. J., and Anselstetter, V. (1977) Hoppe-Seyler’s Z. Physiol. Chem. 3S8, 1287. 11. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250.
BIOCHEMISTRY
96, 130-
138 (1979)
A Precise Method for the Quantitation of Proteins Taking Account Their Amino Acid Composition1
into
HANS-J• ACHIM HORSTMANN lnstitut
ftir
Physiologische D-8520
Chemie, Erlangen,
Universittit Erlangen-Niirnberg, Federal Republic of Germany
Fahrstrasse
17#
Received September 27, 1978 A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-4 solutions containing 1 to 100 pg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of. F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per ahquot of hydrolysate analyzed can be determined.
Various methods have been used for the determination of proteins in biological materials. The well-known calorimetric method developed by Hsien Wu in 1922 (1) and elaborated by Lowry and co-workers in 1951 (2) using the phenol reagent of Folin and Ciocalteu (3) is by far the most frequently used. There are other methods for protein measurement such as estimation of Kjeldahl nitrogen, the determination of peptide groups using the biuret reaction, or the determination of protein-bound dye. By using these methods, however, correct results can only be obtained if the amino acid composition of the protein taken as the reference for calibrating the method corresponds well with that composition of the protein to be determined. Since the 1 Presented in part at the Joint Autumn Meeting of the Biochemical Societies of the German Federal Republic, Switzerland and France, Freiburg, Germany, October Z-5, 1977, and an abstract has appeared in Hoppe-Seyler’s 2. Physiol. Chem. (1977) 358,
i222.
0003-2697/79/090130-09$02.00/O Copyright 10 1979 by Academic FWss, k, All rights of reproduction in any form reserved.
molecular weights of the amino acid residues composing a protein vary from 57 (glytine) to 186 (tryptophan), the true protein content of a biological sample can only be determined by a quantitative analysis of the amino acids released after total hydrolysis taking into consideration their different contributions to the total weight. As a result, I here present a method based on the ninhydrin reaction of hydrolysates of proteins. Since, with the exception of proline and hydroxyproline, the response at 570 nm of all amino acids occurring in proteins to the ninhydrin reagent is virtually the same (4),* the total concentration of amino acids in a hydrolysate can be determined without fractionation of the mixture. The protein mass in the sample is then calculated by multiplying the nanomoles of total amino acids found by a conversion factor corresponding to the z This is also valid for cystine, found in hydrotysates instead of cysteine. 130
PRECISE
QUANTITATION
average molecular weight of 1 nmol of the mixture of the amino acid residues released from the protein. This conversion factor has to be determined. In routine analyses of the same biological material, e.g., erythrocyte membranes, this can be done by quantitative amino acid analysis of the hydrolysate following standard procedures. In the case of pure proteins the conversion factor can be calculated from the amino acid composition reported in the literature. For the majority of proteins the factor lies between 0.11 and 0.12. MATERIALS
AND METHODS
Reagents
All chemicals were of analytical grade. They were purchased from E. Merck, Darmstadt: 9.5 M HCl, suprapure; NaOH; sodium acetate, anhydrous; Tic&, approx 15% solution; sodium citrate, tryst 2HZO; ethylene glycol monomethyl ether, and phenol. The standard amino acid calibration mixture for protein hydrolysates was from Bio-Rad Laboratories, Mtinchen (stock solution: 2.500 ? 0.004 pmol per milliliter of each component; cystine is considered as half-cysteine). Water was deionized. Proteins
Bovine serum albumin, chromatographically pure, was from Boehringer-Mannheim. Protamine sulfate, pure, was obtained from Serva, Heidelberg. Erythrocyte membranes were prepared from freshly drawn human blood (5). Trabecular tissue from human eyes was a gift from Dr. K. Sames, Anatomisches Institut, Erlangen. The tissue was repeatedly extracted with 1 M NaCl at pH 3 in order to remove soluble materials. Solutions
Sodium citrate buffer, 0.2 M, pH 3.2, containing 0.1% phenol. Ninhydrin reagent:
OF PROTEIN
131
Dissolve 25 g of ninhydrin in a solution containing 750 ml of ethylene glycol monomethyl ether, 375 ml of 4 M sodium acetate buffer (pH 5.51), 7.5 g of sodium citrate, and 375 ml of water. Filter the solution into the reagent flask of the amino acid analyzer and add 7.5 ml of a solution of about 15% Tic&. During these procedures the solution is gassed with nitrogen. After standing for at least 6 h the reagent is ready for use. Procedures Step 1: Hydrolysis of proteins. Fill 5 to 20 ~1 of sample solution or suspension containing 1 to 100 pg protein into fine capillary tubes as used for the determination of blood sugar (internal diameter 0.8 to 1.2 mm, not heparinized; Braun , Melsungen) . In order to determine the transferred volumes exactly, measure the length of the liquid column and the internal diameter of the capillary with utmost accuracy, using a measuring microscope (E. Leitz, Wetzlar), or weigh the filled capillary on a microbalance. Fill 1.4 times this volume of 9.5 M HCl into the capillary tube and fuse both its ends. Mix the contents by shaking. Hydrolyse for 24 h at 105’C with occasional mixing. Force the solution into one end of the capillary tube and cut the glass just above the meniscus. Transfer the hydrolysate into a small plastic vessel using a long-tipped capillary pipet operated by mouth. Rinse both ends of the capillary repeatedly with a total volume of 200 ~1 of water. In order to hydrolyze dry materials weigh from 0.3 to 2 mg of the sample into small glass vessels (7.5 x 70 mm), add 200 ~1 of 5.5 M HCI, gas with nitrogen, fuse the vessel, and heat in an oven for 24 h at 105C. Step 2: Removal of ammonia by d$fusion.
Add about 1.5 times the volume of 9.5 M HCl, as used in step 1, of 10 M NaOH to the hydrolysate and mix it. The final pH
132
HANS-JOACHIM
should be about 10. Place the vessel in an almost flat position into a desiccator containing 1 M sulfuric acid and evacuate until a pressure of 40 mm Hg is reached. The diffusion of ammonia is allowed to proceed at room temperature for at least 5 h. Then acidify the hydrolysate with a calculated volume of 9.5 M HCl and desiccate the contents to dryness in a desiccator containing PZOSand pellets of KOH. Step 3: Determination
of ninhydrin
color.
Dissolve the dry residue from step 2 in an appropriate volume (e.g., 50 to 500 ~1) of 0.2 M sodium citrate buffer, pH 3.2. The convenient final concentration of total amino acids is about 1 nmol per microliter, corresponding to about 0.1 pg of protein per microliter. The total amino acids are quantitated without fractionation using the continuousflow system of an amino acid analyzer (e.g., BioCal 200). In order to achieve this, the apparatus is quickly modified by mounting the sample injection block between the outlet of the resin column and the ninhydrin mixing chamber. The flow of the buffer is adjusted to 35 ml/h, and the flow of the ninhydrin reagent to 25 ml/h. The temperature of the resin column is 65’C. By this means the pressure waves caused by the buffer pump are smoothed down to zero before they arrive at the injection block. Inject 2 to 20 ~1 of the solution to be tested containing up to 15 nmol of amino acids into the running buffer stream via the sample injection block using a calibrated microsyringe. Injecting should be finished within 10 s and can be repeated every 5 min. Calibrate the ninhydrin reaction by injecting aliquots of 2 to 10 ~1 of the standard amino acid calibration mixture, containing 0.85 nmol of total amino acids in 1.0 ~1.~ z A mixture of equal amounts of the 17 amino acids found in acid hydrolysates simulates a hydrolysate of a protein better than a solution of one pure amino acid, e.g., leucine, since the slightly varying color yields obtained from the different amino acids are balanced out.
HORSTMANN TABLE
1
WEIGHT OF 1 nmol OF RESIDUE OF AMINO ACID FOUND IN HYDROLYSATES OF PROTEINS
Amino acid residue
i
1 2 3 4
Asp Thr Ser
b
bG3) 0.115 0.101 0.087
GIU
0.129
5
ROa
6 7 8
Ala
0.097 0.057 0.071
9 10 11 12 13 14 15 16 17 I8 19 20
GUY CYS
Val Met Be Leu Vr Phe LYS His A@ TV Asn Gin
0.103 0.099 0.131 0.113 0.113 0.163 0.147 0.128 0.138 0.154 0.186 0.114 0.128
a Hydroxyproline (b = 0.113) should be treated like proline by calculating F.
The absorbance is measured at 570 nm using a microcuvette with 14-mm light path. Prior to use all the ammonia from the calibration mixture has to be removed as given in step 2. Determine, e.g., by integration, the areas under the peaks resulting from the ninhydrin reaction of the unfractionated mixture of amino acids. The peak areas obtained from the calibration mixture are used to establish a standard curve by plotting the areas versus the nanomoles of amino acids injected. Preferably, least-mean-squares regression is calculated from these values using a programmable desk-top calculator. With the parameters for a straight-line relationship thus determined, the nanomoles of amino acids of the sample can then be computed directly from the observed areas. Step 4: Calculation
of the protein
mass.
The weight of a protein is the sum of the
PRECISE QUANTITATION
FIG. I. Diagram of analysis of total amino acids. Aliquots injected repeatedly. For further conditions see text.
weights of the amino acid residues the protein is composed of. If the amino acid composition is known, the mass of the protein can be calculated by multiplying the number of the nanomoles of total amino acids found by the average molecular weight (in pg) of the residues of that amino acid mixture. This average residue weight is called the weight equivalent (WE) and can be calculated as follows: WE = z (Ut.bi)y i=l
[II
where u is the mole fraction of amino acid i per mole of total amino acid composition and b the weight (in pg) of 1 nmol of this respective amino acid residue. Index i represents the at most 20 different amino acids normally occurring in proteins. Their b values are given in Table 1. However, during hydrolysis of a protein performed in HCl tryptophan is destroyed,
133
OF PROTEIN
of samples indicated
have been
cysteine is replaced by cystine, and asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively. Moreover, proline cannot be determined at 570 nm with the ninhydrin reaction applied in the method described. For this reason, a conversion factor F, derived from WE, must be used for the calculation of the protein mass in the analyzed sample. F is characteristic of a protein or a defined protein mixture (e.g., serum or cell organelles) and has to be determined once for all the following quantitations of the same material by quantitative amino acid analysis of an aliquot of the material according to the formula
PI
where c are the nanomoles
of specific
134
HANS-JOACHIM
amino acid i found in the analyzed aliquot and b is the weight (in pg) of 1 nmol of amino acid residue i (see Table l), If solutions of pure proteins of known amino acid composition (including tryptophan) are to be determined, F can be approximated to the value found experimentally after a hydrolysis time of 24 h according to the formula
where u is the mole fraction of the specific amino acid i per mole of total amino acid composition; for i and b see Table I. This relationship was validated for bovine serum albumin. Its F value, 0.1205, calculated from the known amino acid composition (6) according to formula [3], corresponds closely to the F value found experimentally, 0.121, using formula [2]. The protein content of the sample, analyzed according to steps 1 to 3, can now be calculated by multiplying F by the nanomoles of total amino acids found. RESULTS
Reproducibility
and Sensitivity
The peaks obtained by injection of identical aliquots of amino acid solutions are highly reproducible (Fig. 1). Plotting the areas of the peaks of the calibration mixture shown in the figure versus their corresponding amounts of total amino acids resulted in a linear regression line (y = 0.076~ - 0.027; r = 0.999). Using this equation the amino acid concentration of the hydrolysate of the human erythrocyte membranes injected was calculated from the peak areas obtained (X values) to 5.33 ? 0.06 nmol/ 10 ~1 (mean * SD). The coefficient of variation was 1.1% (n = 6). Figure 2 illustrates a standard curve obtained from 20 injections of different volumes of the calibration mixture. The response is linear up to 17 nmol of the amino
HORSTMANN
acids tested. The means and standard deviations for 12 calibration runs made on different days were: correlation coefficient = 0.996 ? 0.005; intercept = 0.004 k 0.023; slope = 0.073 2 0.004. In a typical assay, a sample of a hydrolysate of unknown amino acid concentration is escorted by at least two different volumes of the calibration mixture and the aliquots are injected five times each. The method measures amino acid concentrations as low as 0.3 nmol/lO ~1, corresponding to a protein concentration of approximately 0.03 pg/lO ~1 hydrolysate injected. Variability
of Conversion Factor F
As evidenced by Fig. 3 the conversion factor F varies considerably according to the amino acid composition of a protein. For most proteins F lies between 0.100 and 0.125. Extreme values are related to elastin (0.083) and to the protamines (e.g., salmin 0.147), respectively. The assay has been tested with two pure proteins and two defined biological materials selected for their divergent amino acid composition. Compared to the weight equivalent WE calculated according to formula [1] from the amino acid composition found (see Table 2), F is always of a higher value conditioned by the different proline content of the proteins investigated. Reliability
of the Method
The only reliable method for the determination of the amount of protein in a biological material is to sum up the weights of the amino acid residues found in the protein by quantitative amino acid analysis. Bearing in mind this basic condition I tested the reliability of the new method by dividing the hydrolysates of the selected proteins shown in Table 2 into two parts, A and B. Part A was analyzed by fractionation using an automatic amino acid analyzer and the amount of protein present in the
PRECISE QUANTITATION
AMINO
ACID
OF PROTEIN
135
(ramok)
FIG. 2. Standard curve obtained with the assay, using an amino acid calib~tion mixture of known concentration. The area under a peak is calculated by multiplying the maximum AAs7@nm of a peak by its width (mm) at half-maximum absorbance. The peak areas obtained are plotted versus the nanomoles of total amino acids applied. Each point results from three to four injections of the same amino acid amount. All values are within the circle diameters. The linear regression line is inserted.
sample was calculated by summing up the weights of all the amino acid residues found. F was calculated according to formula [Z] . Part B was treated as described under Materials and Methods, steps 2 to 3, The amount of protein was calculated by multiplying the nanomoles of total amino acids found at 570 nm in the u~ractionated mixture by the conversion factor F derived from the respective part A. Figure 4 illustrates the results of the comparison of the two methods of protein determination. Taking the protein amounts found in the respective parts A by quantitative amino
acid analysis as the actual values, a linear regression line for all B analyses (n = 86) can be calculated: y = 0.97% + 0.032; r = 0.982. The overall recovery of protein, irrespective of its amino acid composition, determined by the new method was 1.017 ztz0.046. Comparison
with Other Methods
The protein of different erythrocyte membrane preparations was also assayed by two other procedures, (i) the Lowry method, as modified by Wang and Smith (8) and, (ii) the Biuret method according to Futterman
136
HANS-JOACHIM
HORSTMANN
TABLE AMINO
ACID COMPOSITION, WEIGHT EQUIVALENT WE, AND CONVERSION PROTEINS OR PROTEIN MIXTURES TO TEST THE PROTEIN ASSAY
Amino acid ASP Thr Ser GhI Pro GlY Ala CYS Val Met Be Leu Tyr Phe LYS His kz HYP WP Q4 F @td
2
Trabecular tissuea
Erythrocyte membranea
7.0 3.8 5.8 11.1 8.7 20.5 9.4
9.2 5.4 7.2 13.8 5.6 6.7 8.1
5.4
6.9 1.7 4.7 12.8 2.1 4.4 4.8 2.3 4.4
3.3 6.7 2.2 2.9 4.3 1.1 4.6 3.0 0.100 0.113
Bovine serum aIbumin”
0.113 0.121
a Moles per 100 mol of amino acid composition. b WE and F were calculated from the amino acid composition formulas 1 and 2, respectively.
DISCUSSION
The method presented is especially suited for insoluble biological materials such as cell
Protaminea
9.9 6.1 4.8 14.2 6.3 2.8 8.2 3.8 5.8 0.4 2.0 10.2 3.0 4.5 11.4 2.8 3.7
0.110 0.116
and Rollins (9), each time using bovine serum albumin as the standard. Referred to the protein content of these samples calculated from total amino acid analysis, only about 0.8 was found with the Lowry method and only about 0.5 with the Biuret method, respectively (IO). These findings show that using serum albumin as the reference protein makes it impossible to quantitate the protein of the erythrocyte membrane by these two methods. Notice that the weight equivalents of serum albumin and of the membrane protein mixture are very similar and, also, that their amino acid compositions are not strikingly different (see Table 2).
FACTOR F, FOR FOUR DESCRIBED
found in the hydrolysates
6.9 12.0 5.0 1.0 3.6 0.8
70.6 0.134 0.153 according to
organelles and for tissue probes available in small amounts. For the precise determination of the protein content of these samples only 5 to IO ~1 of suspension or solution containing as little as 100 ng of protein are needed. This procedure has been successfully applied by us to comparative studies of the peptide patterns obtained by enzymatic digestion of isolated membrane proteins. Using this technique it is necessary to know exactly both the ratio of protease to protein and the amounts of the peptides loaded onto polyacrylamide gradient gels.4 During hydrolysis of proteins asparagine and glutamine are split into ammonia and aspartic acid and glutamic acid, respectively. By this, however, the weights of these residues remain practically unchanged d Anselstetter, V. (1979) J. Chromafogr.,
172, 49-56.
PRECISE QUANTITATION
137
OF PROTEIN
Free amino acids, oligopeptides, amino sugars, and other soluble substances yielding color at 570 nm with ninhydrin interfere with this protein determination. For this reason, these substances must be removed from the sample prior to hydrolysis. Cell organelles and tissue probes should be washed repeatedly by suspension in a suitable buffer and collected by centrifugation. Soluble proteins can be separated from interfering low-molecular-weight material by trichloroacetic acid precipitation, using the method of Bensadoun and Weinstein (11) in the case of very dilute samples. Besides ninhydrin other reagents for the determination of a-amino groups may be used provided that all amino acids reacting with the reagent give the same color response. This may be the case for fluorescamine (Hoffmann-La Roche). In this way the
r
e 0.06
k 0.10
ha]
1 c 0 12
P
014
FIG. 3. Distribution of the conversion factor F of 93 different proteins. F is the weight (in pg) of 1 nmol of the mixture of amino acid residues composing a specific protein. The amino acid composition for most of the proteins selected were taken from the data compilated by Kirschenbaum [e.g., ref. (7)] using the formula [3] for the calculation of E. The positions of some proteins are indicated: a = bovine serum albumin; c = casein: e = elastin (bovine aorta); h = hemoglobin; k = collagen; p = protamine.
(see Table 1). Cysteic acid obtained by oxidation of cysteine residues should be taken as cysteine when calculating F. An amino acid analyzer slightly modified as described is used for the assay, but should not be a prerequisite. Any spectrophotometer adapted to measure a small volume (e.g., 0.1 ml) in a cuvette with a long light path should be suitable as well. However, for the determination of the conversion factor F an amino acid analyzer is needed unless F is otherwise known.
PROTEIN (pg),
method
A
FIG. 4. Comparison of two different methods for the determination of protein. Method A: protein found by quantitative amino acid analysis; method B: protein found by the assay described. The linear regression line is inserted. For further information see text. (0) Trabecular tissue, human eye; (0) human erythrocyte membrane; (A) bovine serum albumin; (A) protamine. Each symbol represents the average of two to six injections of the same ahquot of hydrolysate. Vertical lines indicate the range when these values are larger than the symbol diameters.
138
HANS-JOACHIM
sensitivity of the assay may be increased considerably. ACKNOWLEDGMENTS I wish to express my gratitude to Dr. Volker Anselstetter for helpful discussions and critical commerits. This work was supported by a grant (Ho 118/l 1) from the Deutsche Forschungsgemeinschaft.
REFERENCES 1. Wu, H. (1922)J. Bioi. Chem. Sl, 33-39. 2. Lowry, 0. H., Rosebrough, H. I., Farr, A. L., and Randall, R. I. (1951) J. BioL Chem. 193, 265-275. 3. Folin, O., and Ciocalteu, V. (1927) J. Bio/. Chem. 73, 627-650.
HORSTMANN
4. Moore, (Them.
S., and Stein, W. H. (1954) J. Biol. 211,907-913.
5. Anselstetter, V., and Horstmann, H. J. (1975) Eur. J. Biochem. S6, 259-269. 6. Spahr, P. F., and Edsall, J. T. (1964) J. Biol. Chem. 239, 850-854. 7. Kirschenbaum, D. M. (1977) Anal. Biochem. 83, 521-550. 8. Wang, C. S., and Smith, R. L. (1975) Anal. Biochem. 63, 414-417. 9. Futterman, S., and Rollins, M. H. (1973) Ana/. Biochem. Sl, 443-447. 10. Stierstorfer, S., Horstmann, H. J., and Anselstetter, V. (1977) Hoppe-Seyler’s Z. Physiol. Chem. 3S8, 1287. 11. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250.
