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Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal
ANALYTICAL
BIOCHEMISTRY
111,
Calorimetric
R. BRYAN Department
220-226 (1981)
Determination of Arginine by p-Nitrophenylglyoxall YAMASAKI,
of Food
Science
DOROTHY
A.
and Technology, Received
SHIMER,
University August
Residues
AND
ROBERT
of California,
Davis,
in Proteins
E. FEENEY~ California
95616
15, 1980
A new method is presented for the calorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 M sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer’s law in the range 0.03-0.33 mM arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.
The majority of published methods for the direct calorimetric determination of arginine residues in proteins have largely been based on the Sakaguchi reaction (1). Many modifications of the original technique have been developed because of the instability of the reaction products and the high background produced (2,3). In spite of these modifications, however, accuracy and reproducibility still appear marginal at best due to the extreme critical handling of the reagents. Nitromalondialdehyde sodium salt has been reported to react with arginine under strongly alkaline conditions to form a product whose absorption maximum is found near 335 nm (4). Nitromalondialdehyde sodium salt had been used to modify the single arginine residue in S-carboxymethyl B chain ’ This work was supported in part by National Institutes of Health Grants HL 18619 and AM 26031. This material constitutes part of the thesis of R.B.Y. submitted to the Graduate Division of the University of California at Davis in partial fuhillment of the requirements for a Ph.D. degree in Biochemistry. Portions of this work were presented at the meeting of the Federation of American Societies for Experimental Biology, April 13-18, 1980, Anaheim, Calif. 2 To whom correspondence should be addressed. 0003-2697/81/040220-07$02.00/O Copyright AU rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
220
of insulin (4). It should be noted that the sodium salt of nitromalondialdehyde is impact sensitive and thermally unstable and should be handled as a potentially explosive material (5). The reaction of 9, lo-phenanthrenedione with monosubstituted guanidines reportedly yields a fluorescent product and is the basis for a number of fluorometric methods for the detection and determination of arginine and arginine-containing proteins (6-8). The reactions generally take place in strongly alkaline water-ethanol (6-8). Recently, Borders et al. (9) reported the use of 4-hydroxy-3-nitrophenylglyoxal as a chromophoric reagent for arginine residues in creatine kinase. The mild conditions used make 4-hydroxy-3-nitrophenylglyoxal attractive for work requiring nondenaturing conditions.p-Hydroxyphenylglyoxal, another chromophoric reagent for arginine residues (lo), was also found suitable for protein work where nondenaturing conditions are desired. Both reagents undergo only slight spectral changes upon reaction with arginine so that unreacted reagent must be removed before measuring the absorbance of proteinbound chromophore.
ARGININE
RESIDUE
DETERMINATION
BY p-NITROPHENYLGLYOXAL
221
(color stabilizing agent) concentration, pH, p-nitrophenylglyoxal concentration, and temperature on the color reaction ofp-nitrophenylglyoxal with arginine were studied in order to optimize the conditions for a new calorimetric method for the determination of arginine. Each parameter was varied while holding the remaining parameters constant under the following general conditions: To 3.00 ml of 0.25 mM L-arginine in 0.1 M sodium pyrophosphate/O. 15 M sodium asEXPERIMENTAL PROCEDURES corbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. The reacMaterials tion was allowed to proceed at 23°C and at p-Nitrophenylglyoxal was prepared from intervals the absorbance at 475 nm was read p-nitroacetophenone (Aldrich) by the method against a blank containing no arginine. of Steinbach and Becker (11). Glutathione The specificity of the color reaction for (reduced) and a-amino acids were pur- arginine was tested by substituting other chased from Nutritional Biochemicals Corcompounds at the same concentration used poration. N *- Acetylhistidine, l-methylhisfor arginine and reading the absorbances at tidine, and 3-methylhistidine were obtained 475 nm after 30 min at 30°C. from Calbiochem.3 Imidazole, IV”-acetylDetermination ofarginine residues inproarginine, arginine methyl ester, 2-mercaptoteins. The following reagents were prepared: ethanol, bovine serum albumin, bovine (A) 0.1 M sodium pyrophosphatelo. 15 M sopancreatic ribonuclease A, rabbit muscle dium ascorbate, pH 9.0 (prepared fresh daily creatine phosphokinase, and yeast alcohol using nitrogen-purged water); (B) 10% pdehydrogenase were purchased from Sigma. nitrophenylglyoxal in methanol (prepared Bovine trypsin and diaphorase (Clostridiurn fresh daily);4 (C) 0.1% subtilisin in 0.01 M kluyveri) were obtained from Worthington. sodium phosphate, pH 7.9; (D) 0.1% bovine Subtilisin (Novo) was obtained from Novo trypsin in 0.001 M HCVO.02 M CaCl,. Industries. Arginyl-antifreeze glycopeptide To 0.20 ml of sample containing an equiva8 was prepared as previously described (10). lent of O.l- 1.O pm01 arginine (for proteins, Chicken ovotransferrin was isolated from the typically l-2 mg) in deionized water or 0.1 M egg white of chicken (Gab gal/us) accordsodium pyrophosphate, pH 9.0, were added ing to the method of Rogers et ul. (12). Crys40 ~1 of(C) and 40 ~1 of(D). After incubating talline chicken ovalbumin was prepared ac- at 37°C for 3 h, the solution was cooled and cording to the method of Kekwick and made up to 3.00 ml with (A). Color developCannan (13) and recrystallized twice. Pen- ment was initiated by mixing in 25 ~1 of reguin ovalbumin was prepared according to agent (B). The reaction mixture was incuthe method of Ho et al. (14). All other matebated at 30°C for 30 min and subsequently rials used were purchased as reagent grade. cooled below 20°C in an ice bath before measuring the absorbance at 475 nm against Methods a reagent blank containing the correspondReaction of p-nitrophenylglyoxal with ing amounts of (C) and (D). The arginine arginine. The effects of sodium ascorbate content of the sample was then determined
This paper describes a new method for the calorimetric determination of arginine residues in proteins by the use of p-nitrophenylglyoxal. The procedure requires no critical handling of reagents and is performed under mild conditions. There is no need to remove excess reagent before measuring the color produced because the absorption maximum of the product is greatly different from that of the reagent.
S Throughout this paper the imidazole ring of histidine and its derivatives is numbered according to IUPAC nomenclature.
4 p-Nitrophenylglyoxal should be stored desiccated in the dark at O-ST. Samples stored under these conditions were found to be stable for at least 18 months.
222
YAMASAKI,
SHIMER,
by comparison with a standard curve prepared using a stock solution of arginine of known concentration. When preparing a standard curve using free arginine or when assaying for arginine residues in intact proteins, the additions of (C) and (D) and the incubation step at 37°C for 3 h were omitted. All spectrophotometric measurements were performed using a Beckman Model 35 recording spectrophotometer. Protein concentrations were determined by measurement of their absorbance at 280 nm in deionized water or 10 mM sodium phosphate buffer, pH 7.0, using the following absorption coefficients (E:&): chicken ovotransferrin, 11.6 (15); bovine serum albumin, 6.60 (16); bovine pancreatic ribonuclease A, 6.95 (17); chicken ovalbumin, 7.50 (15); rabbit muscle creatine phosphokinase, 8.76 ( 18); yeast alcohol dehydrogenase, 12.6 (19). Concentrations of penguin ovalbumin and diaphorase solutions were determined by dry weight. Concentrations of arginyl-antifreeze glycopeptide 8 solutions were determined by amino acid analysis. Complete acid hydrolysis of protein samples was achieved with 6 M HCl at 110°C for 22 h in evacuated sealed tubes. Hydrolysates were analyzed on a Technicon auto analyzer. RESULTS Reaction of p-Nitrophenylglyoxal with Arginine
p-Nitrophenylglyoxal in 0.1 M sodium pyrophosphate, pH 9.0, was found to absorb maximally at 265 nm with a molar absorption coefficient of 8.1 x 103 M-l cm-l. Reaction of p-nitrophenylglyoxal with arginine at mildly alkaline pH resulted in the formation of a colored product whose absorbance maximum was found at 450 nm (Fig. 1). Constant purging of nitrogen gas into the reaction mixture allowed the orangered color to develop and remain. Shaking the colored reaction mixture in the presence of ordinary air caused a rapid fading of the
AND FEENEY
I.., ‘......, %. ‘.. \
400
500 WAVELENGTH
600 (nm)
FIG. 1. Absorption spectra of the arginine-p-nitrophenylglyoxal reaction mixture. To 3.00 ml of 0.25 mM t-arginine in 0.1 M sodium pyrophosphate/O. 15 M sodium ascorbate, pH 9.0, was added 25 pi of 10% p-nitrophenylglyoxal in methanol (4.7 mM p-nitrophenylglyoxal in the reaction mixture). The absorbance scans were recorded after 70 min at 23°C. The concentration of p-nitrophenylglyoxal was approximately 20 times the concentration of arginine. Reaction mixture vs water (. .), reagent blank vs water (-), reaction mixture vs reagent blank (-).
color. Sodium ascorbate, at concentrations greater than 0.10 M, stabilized the color enough to permit the application of this reaction. Figure 2 shows the effect of sodium ascorbate concentration on color development. A concentration of 0.15 M sodium ascorbate was regarded as the best concentration when the color development in the blank was taken into account. The absorbance was routinely measured at 475 nm because of the high blank encountered at 450 nm (Fig. 1) in the presence of a 20-fold molar excess ofp-nitrophenylglyoxal. Ordinary room light appeared to have no effect on the color development. Hydroquinone, sodium sulfite, sodium borohydride, 3,5dihydroxybenzoic acid, sodium hydrosulfite, thiodiglycol, and sodium arsenite were ineffective in stabilizing the color. In general, the rate of color development increa‘sed with pH, p-nitrophenylglyoxal concentration, and temperature. The color
ARGININE
cl REACTION
RESIDUE DETERMINATION
50 TIME (min)
100
FIG. 2. Effect of sodium ascorbate concentration on color development. To 3.00 ml of 0.25 mM L-arginine in 0.1 M sodium pyrophosphate, pH 9.0, containing sodium ascorbate (concentration given below), was added 25 ~1 of lO%p-nitrophenylglyoxal in methanol. The reaction was allowed to proceed at 23°C. The concentrations are: 0.05 M sodium ascorbate (A), 0.10 M sodium ascorbate (0), 0.15 M sodium ascorbate (A), 0.20M sodium ascorbate (0).
appeared unstable at pH 9.5 and above, even in the presence of 0.15 M sodium ascorbate. At pH 10.0 and above, the blank, as well as the test solution, became turbid. For routine work pH 9.0 was therefore regarded as optimal. Aliquots of reagent (B) (10% methanolic g-nitrophenylglyoxal) larger than 25 ,ul led to turbidity in reaction volumes of 3.00 ml or less. For 3.00-ml reaction volumes, 25 ~1 of (B) gave the highest rate of color development without leading to turbidity. At 23°C color development was complete after 70 min and remained stable for an additional 30 min. By increasing the temperature to 3O”C, the reaction time was shortened to 30 min, but the color slowly faded about 10 min after reaching complete color development. Cooling (in an ice bath) below 20°C after achieving complete color development prevented this problem. Higher temperatures were unsuitable as the color was found too unstable to be of any value. Other compounds were tested for possible interfering absorbances that might occur as
223
BY p-NITROPHENYLGLYOXAL
a result of side reactions. All 20 common amino acids found in proteins, as well as arginine methyl ester, cystine, cysteic acid, methionine sulfone, methionine sulfoxide, 2-mercaptoethanol, glutathione (reduced), sulfosalicylic acid, cystine dimethyl ester, dithiodiglycolic acid, urea, guanidine, sodium dodecyl sulfate, imidazole, l-methylhistidine, 3-methylhistidine, and the N”acetyl derivatives of arginine and histidine were individually tested at the same concentration (0.25 mM). Possible interfering compounds and pertinent related ones are shown in Table 1. All others tested but not listed in Table 1 gave absorbances of less than 0.005 at 475 nm. Free histidine reacted to give an absorbance -24% of that for arginine. It appeared that the free a-amino group must be present for this side reaction to occur since neither N”-acetylhistidine nor imidazole reacted to give an interfering absorbance. The imidazole ring must also play a role since 1-methylhistidine interfered TABLE ABSORPTION WITH
OF VARIOUS
1 COMPOUNDS
TREATED
p-NITROPHENYLGLYOXAL
Compound Arginine N”-Acetylarginine Arginine methyl ester Histidine N”Acetylhistidine Imidazole I-Methylhistidine 3-Methylhistidine N”-Acetyl-l-methylhistidine N”-Acetyl-3-methylhistidine Cysteine Glutathione (reduced) 2-Mercaptoethanol
Absorbance at 475 nm” 0.715 0.700 0.720 0.168 0 0 0.235 0 0.002 0 0.020 0.019 0.026
‘I To 3.00 ml of a 0.25 mM solution of the test compound in 0.1 M sodium pyrophosphate/O.lS M sodium ascorbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. After incubating for 30 min at 30°C. the solutions were cooled in an ice bath below 20°C and the absorbances at 475 nm were read against a reagent blank in I .O-cm cuvettes.
YAMASAKI,
0
0.2
ARGININE
CONCENTRATION
SHIMER,
0.4
(mM)
FIG. 3. Standard curve for arginine. To 3.00 ml of 0.03-0.33 mM L-arginine in 0.1 M sodium pyrophosphate/0.15 M sodium ascorbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. The reaction was carried out at 30°C for 30 min before reading the absorption at 475 nm against a reagent blank.
whereas 3-methylhistidine did not. Interference by free histidine and possibly amino terminal histidine in peptides may be prevented by acetylating the free amino group with acetic anhydride. At pH 8 complete acetylation of alanine is accomplished within 5 min with a twofold excess of acetic anhydride (20). Complete acetylation of histidine was obtained in less than 1 min at pH 9.0. Acetylation of 1-methylhistidine in this manner prevented the reaction which led to an interfering absorbance. Free sulfhydryl groups reacted with p-nitrophenylglyoxal, but the resulting absorbance at 475 nm was less than 3% of the absorbance yielded by arginine . Determination of Arginine Residues in Proteins
The methods described under Experimental Procedures were used to determine the number of arginine residues in several proteins of known amino acid composition. Beer’s law was obeyed in the range 0.030.33 mM arginine as shown by the standard curve in Fig. 3. Where intact proteins were
AND FEENEY
assayed, the time for complete color development (to account for all of the arginine residues) varied with the protein. The time for complete color development with intact proteins was, in general, much longer than that with free arginine. For a more rapid quantitation of total arginine residues, best results were obtained when protein samples were predigested with a mixture of trypsin and subtilisin. It was found that 3-h digestion time was sufficient for the proteins studied. Table 2 compares the results of the total arginine determination of several proteins (via treatment with trypsin and subtilisin) with the corresponding known arginine contents. Samples treated with trypsin alone gave results intermediate of the results given by the two methods above. Arginine modified with p-nitrophenylglyoxal was found unstable to the conditions for the acid hydrolysis of proteins (-22% regeneration of arginine) and, therefore, the extent of modification as measured by spectrophotometry and amino acid analysis could not be compared. TABLE
2
ESTIMATIONOFTHENUMBEROFARGININERESIDUES PER MOLE OF PROTEIN No. of arginine
residues/ mol of protein”
p-Nitrophenylglyoxal method”
Protein Chicken ovotransferrin Bovine serum albumin Ribonuclease A Chicken ovalbumin Penguin ovalbumin Diaphorase Creatine phosphokinase Yeast alcohol dehydrogenase Arginylantifreeze glycopepfide
8
32.6 24.6 3.9 15.0 7.3 6.5 35.7 34.6 1.W
Expected’ 33.0 24.0 4.0 15.7’ 7.IP 6.6 35.1 31.7 1.0,
” Estimated on the basis of the following molecular weights: chicken ovotransfenin, 76,600 (21); bovine serum albumin, 67.OOil (22); ribonuclease A, 13.683 (23): chicken ovalbumin. 46.000 (15): penguin ovalbumin. 48.000 (24); diaphorase. 24,000 125); creatine phosphokinase. 82.600 (26): yeast alcohol dehydrogenase, 150,ooO (27): argmylantifreeze glycopeptide 8, 2800 t 10,281. 0 Protein samples were digested with a mixture of trypsin and subtilisin prior to assaying for nrginines (see Experimental Procedures). ’ On the basis of the known sequence and/or composition (see above references). n Obtained using the intact glycopeptide. * Obtained by amino acid analysis in our laboratory.
ARGININE
RESIDUE
DETERMINATION
Heat denaturation, alkali treatment, reduction and alkylation of disulfide bonds, and performic acid treatment were used in an attempt to disrupt the native structure of the protein and to fully expose all arginine residues to the reagent. These methods failed to give suitable results due to frequent precipitation of the treated samples. High concentrations of urea (4-8 M) were found unsuitable as they formed colorless solutions with p-nitrophenylglyoxal. Denaturation by 1% sodium dodecyl sulfate led to rates of color formation lower than those with intact proteins. Borate and 2-amino2-(hydroxymethyl)1,3-propanediol buffers could not be used as they severely reduced color formation. Reactions of phenylglyoxal (a homolog of p-nitrophenylglyoxal) with borate (29) and with 2-amino-2-(hydroxymethyl)-1,3-propanediol (30) have been reported. DISCUSSION
p-Nitrophenylglyoxal, in the presence of 0.15 M sodium ascorbate, can be used to reliably determine arginine residues in proteins. Since not all of the arginine residues in intact proteins are necessarily reactive, a method for disrupting the native structure was required for the quantitation of total arginine residues. Treatment with a mixture of trypsin and subtilisin gave satisfactory results in the examples studied. Other methods such as heat denaturation could be suitable for some proteins that do not precipitate under treatment. Workers have reported on modified Sakaguchi procedures that may be applicable to unhydrolyzed proteins, but anomalous results have been reported in certain cases (31). Izumi (32) has concluded that the Sakaguchi method is not applicable to the estimation of arginine residues in proteins without prior hydrolysis. Histidine also reacts with p-nitrophenylglyoxal to give a small interfering absorbance, but only if the free a-amino group is present. A possible explanation involves an attack on the imidazole ring by the pro-
BY p-NITROPHENYLGLYOXAL
225
tonated imine formed from the reaction of the a-amino group with the aldehyde group of p-nitrophenylglyoxal. A similar reaction with formaldehyde has been reported (33, 34). In the case of unhydrolyzed proteins, this interference does not usually arise since the a-amino groups of histidine are blocked in the form of peptide linkages. Only an N-terminal histidine residue, if available for reaction, would have the potential for interfering, and even then its contribution to the total absorbance would normally be small. Acetylation should usually be unnecessary when assaying unhydrolyzed proteins. The same argument can be applied to samples digested with trypsin and subtilisin except now there is a variety of N-terminal residues to deal with. However, it appears improbable that an amount of N-terminalhistidine-containing peptides sufficient to cause significant interference would be created. The data in this study seem to support this argument. Only one example in this study, yeast alcohol dehydrogenase, gave a high value for the total number of arginine residues. The procedures presented in this paper for the determination of arginine residues in proteins by p-nitrophenylglyoxal should easily be adopted for automation. One advantage p-nitrophenylglyoxal has over nearly all existing arginine color reagents is the mild conditions used for color development. Many proteins effectively retain their native structure at pH 9.0.p-Nitrophenylglyoxal might possibly be used to obtain information on the relative reactivities of the arginine residues in native proteins directly. It should be noted that a residue located at the surface of a protein or exposed to solvent may still appear unreactive (35,36). When determining arginine in an amino acid pool such as blood serum, where the amount of free histidine is likely to be significant, acetylation must be included.5 This 5 This is done rapidly and easily by acetylating with acetic anhydride (20). Preliminary results using rat blood serum showed 0.27 mM free arginine by p-nitro-
226
YAMASAKI,
SHIMER,
step is quick and simple and should be included in an automated program. It is interesting that upon reaction with arginine, p-nitrophenylglyoxal undergoes a large spectral shift, whereas phenylglyoxal,6 p-hydroxyphenylglyoxal (lo), and 4-hydroxy-3-nitrophenylglyoxal (9) do not. The colored p-nitrophenylglyoxal product has been used for direct determinations of arginines, while the stablep-hydroxyphenylglyoxal product (10) was used for preparations of chemically modified arginines. ACKNOWLEDGMENTS The authors thank David Sherman for preparing p-nitrophenylglyoxal, David T. Osuga for his many helpful discussions, Chris Howland for editorial assistance, and Clara Robison for typing the manuscript.
REFERENCES
5. 6. I. 8. 9.
10. 11.
Sakaguchi, S. (1925)J. Biochem. (Tokyo) $25-31. Micklus, M. J., and Stein, I. M. (1973) Anal. Biothem. 54,545-553. Alexeenko, L. P., and Orekhovich, V. N. (1970) Int. .I. Protein Res. 2, 241-246. Signor, A., Bonora, G. M., Biondi, L., Nisato, D., Marzotto, A., and Scoffone, E. (197l)Biochemistry 10, 2748-2752. Fanta, P. E. (1952) Organ. Syn. 32,95-96. Yamada, S., and Itano, H. A. (1966) Biochim. Biophys. Acta 130, 538-540. Itano, H. A., Hirota, K., Kawasaki, I., and Yamada, S. (1976)Anal. Biochem. 76,134-141. Smith, R. E., and MacQuarrie, R. (1978)AnaI. Biothem. 90, 246-255. Borders, C. L., Jr., Pearson, L. J., McLaughlin, A. E., Gustafson, M. E., Vasiloff, J., An, F. Y., and Morgan, D. J. (1979) Biochim. Biophys. Acta 568, 491-495. Yamasaki, R. B., Vega, A., and Feeney, R. E. (1980) Anal. Biochem. 109, 32-40. Steinbach, L., and Becker, E. I. (1954) J. Amer. Chem. Sot. 76, 5808-5810.
phenylglyoxal while amino acid analysis showed 0.29 mM free arginine after removing serum proteins with sulfosalicylic acid. 6 Phenylglyoxal was found to absorb maximally at 252 nm in water. Phenylglyoxal-modified arginine was reported by Takahashi (37) to absorb maximally at 250 nm in water.
AND
FEENEY
12. Rogers, T. B., Gold, R. A., and Feeney, R. E. (1977) Biochemistry 16,2299-2305. 13. Kekwick, R. A., and Cannan, R. K. (1936) Biothem. J. 30,227-234. 14. Ho, C. Y.-K., Prager, E. M., Wilson, A. C., Osuga, D. T., and Feeney, R. E. (1976) J. Mol. Evol. 8,271-282. 15. Feeney, R. E., and Allison, R. G. (1969) Evolutionary Biochemistry of Proteins. Homologous and Analogous Proteins from Avian Egg Whites, Blood Sera, Milk, and Other Substances, p. 30, Wiley-Interscience, New York. 16. Wetlaufer, D. B. (1962)Advan. Protein. Gem. 17, 303-390. 17. Sherwood, L. M., and Potts, J. T., Jr. (1965) J. Biol. Chem. 240, 3799-3805. 18. Noda, L., Kuby, S. A., and Lardy, H. A. (1954) J. Biol. Chem. 209, 203-210. 19. Hayes, J. E., Jr., and Velick, S. F. (1954) J. Biol. Chem. 207,225-244. 20. Smyth, D. G. (1967) J. Biol. Chem. 242, 15921598. 21. Osuga, D. T., and Feeney, R. E. (1974) in Toxic Constituents of Animal Foodstuffs (Liener, I. E., ed.), pp. 39-71, Academic Press, New York. 22. Putnam, F. W. (1965)in The Proteins (Neurath, H., ed.), 2nd ed., Vol. 3, pp. 153-267, Academic Press, New York. 23. Hirs, C. H. W., Moore, S., and Stein, W. H. (1956) J. Biol. Chem. 219,623-642. 24. Aminlari, M. (1980) Ph.D. Thesis, University of California, Davis, Calif. 25. Kaplan, F., Setlow, P., and Kaplan, N. 0. (1969) Arch. Biochem. Biophys. 132, 91-98. 26. Yue, R. H., Palmieri, R. H., Olson, 0. E., and Kuby, S. A. (1967) Biochemistry 6,3204-3227. 27. Sund, H., and Theorell, H. (1%3) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 7, pp. 25-83, Academic Press, New York. 28. Osuga, D. T., and Feeney, R. E. (1978) J. Biol. Chem. 253,5338-5343. 29. Rogers, T. B., Borresen, T., and Feeney, R. E. (1978) Biochemistry 17, 1105- 1109. 30. Takahashi, K. (1968) J. Biol. Chem. 243, 61716179. 31. Tomlinson, G., and Viswanatha, T. (1974) Anal. Biochem. 60, 15-24. 32. Izumi, Y. (1%5) Anal. Biochem. 12, l-7. 33. Neuberger, A. (1944) Biochem. J. 38, 309-314. 34. Fraenkel-Conrat, H., and Olcott, H. S. (1948) J. Biol. Chem. 174, 827-843. 35. Riordan, J. F. (1973)Biochemistry 12,3915-3923. 36. Patthy, L., and Thesz, J. (1980) Eur. J. B&hem. 105, 387-393.
37. Takahashi, K. (1977) J. Biochem. (Tokyo) 81,403414.
BIOCHEMISTRY
111,
Calorimetric
R. BRYAN Department
220-226 (1981)
Determination of Arginine by p-Nitrophenylglyoxall YAMASAKI,
of Food
Science
DOROTHY
A.
and Technology, Received
SHIMER,
University August
Residues
AND
ROBERT
of California,
Davis,
in Proteins
E. FEENEY~ California
95616
15, 1980
A new method is presented for the calorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 M sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer’s law in the range 0.03-0.33 mM arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.
The majority of published methods for the direct calorimetric determination of arginine residues in proteins have largely been based on the Sakaguchi reaction (1). Many modifications of the original technique have been developed because of the instability of the reaction products and the high background produced (2,3). In spite of these modifications, however, accuracy and reproducibility still appear marginal at best due to the extreme critical handling of the reagents. Nitromalondialdehyde sodium salt has been reported to react with arginine under strongly alkaline conditions to form a product whose absorption maximum is found near 335 nm (4). Nitromalondialdehyde sodium salt had been used to modify the single arginine residue in S-carboxymethyl B chain ’ This work was supported in part by National Institutes of Health Grants HL 18619 and AM 26031. This material constitutes part of the thesis of R.B.Y. submitted to the Graduate Division of the University of California at Davis in partial fuhillment of the requirements for a Ph.D. degree in Biochemistry. Portions of this work were presented at the meeting of the Federation of American Societies for Experimental Biology, April 13-18, 1980, Anaheim, Calif. 2 To whom correspondence should be addressed. 0003-2697/81/040220-07$02.00/O Copyright AU rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
220
of insulin (4). It should be noted that the sodium salt of nitromalondialdehyde is impact sensitive and thermally unstable and should be handled as a potentially explosive material (5). The reaction of 9, lo-phenanthrenedione with monosubstituted guanidines reportedly yields a fluorescent product and is the basis for a number of fluorometric methods for the detection and determination of arginine and arginine-containing proteins (6-8). The reactions generally take place in strongly alkaline water-ethanol (6-8). Recently, Borders et al. (9) reported the use of 4-hydroxy-3-nitrophenylglyoxal as a chromophoric reagent for arginine residues in creatine kinase. The mild conditions used make 4-hydroxy-3-nitrophenylglyoxal attractive for work requiring nondenaturing conditions.p-Hydroxyphenylglyoxal, another chromophoric reagent for arginine residues (lo), was also found suitable for protein work where nondenaturing conditions are desired. Both reagents undergo only slight spectral changes upon reaction with arginine so that unreacted reagent must be removed before measuring the absorbance of proteinbound chromophore.
ARGININE
RESIDUE
DETERMINATION
BY p-NITROPHENYLGLYOXAL
221
(color stabilizing agent) concentration, pH, p-nitrophenylglyoxal concentration, and temperature on the color reaction ofp-nitrophenylglyoxal with arginine were studied in order to optimize the conditions for a new calorimetric method for the determination of arginine. Each parameter was varied while holding the remaining parameters constant under the following general conditions: To 3.00 ml of 0.25 mM L-arginine in 0.1 M sodium pyrophosphate/O. 15 M sodium asEXPERIMENTAL PROCEDURES corbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. The reacMaterials tion was allowed to proceed at 23°C and at p-Nitrophenylglyoxal was prepared from intervals the absorbance at 475 nm was read p-nitroacetophenone (Aldrich) by the method against a blank containing no arginine. of Steinbach and Becker (11). Glutathione The specificity of the color reaction for (reduced) and a-amino acids were pur- arginine was tested by substituting other chased from Nutritional Biochemicals Corcompounds at the same concentration used poration. N *- Acetylhistidine, l-methylhisfor arginine and reading the absorbances at tidine, and 3-methylhistidine were obtained 475 nm after 30 min at 30°C. from Calbiochem.3 Imidazole, IV”-acetylDetermination ofarginine residues inproarginine, arginine methyl ester, 2-mercaptoteins. The following reagents were prepared: ethanol, bovine serum albumin, bovine (A) 0.1 M sodium pyrophosphatelo. 15 M sopancreatic ribonuclease A, rabbit muscle dium ascorbate, pH 9.0 (prepared fresh daily creatine phosphokinase, and yeast alcohol using nitrogen-purged water); (B) 10% pdehydrogenase were purchased from Sigma. nitrophenylglyoxal in methanol (prepared Bovine trypsin and diaphorase (Clostridiurn fresh daily);4 (C) 0.1% subtilisin in 0.01 M kluyveri) were obtained from Worthington. sodium phosphate, pH 7.9; (D) 0.1% bovine Subtilisin (Novo) was obtained from Novo trypsin in 0.001 M HCVO.02 M CaCl,. Industries. Arginyl-antifreeze glycopeptide To 0.20 ml of sample containing an equiva8 was prepared as previously described (10). lent of O.l- 1.O pm01 arginine (for proteins, Chicken ovotransferrin was isolated from the typically l-2 mg) in deionized water or 0.1 M egg white of chicken (Gab gal/us) accordsodium pyrophosphate, pH 9.0, were added ing to the method of Rogers et ul. (12). Crys40 ~1 of(C) and 40 ~1 of(D). After incubating talline chicken ovalbumin was prepared ac- at 37°C for 3 h, the solution was cooled and cording to the method of Kekwick and made up to 3.00 ml with (A). Color developCannan (13) and recrystallized twice. Pen- ment was initiated by mixing in 25 ~1 of reguin ovalbumin was prepared according to agent (B). The reaction mixture was incuthe method of Ho et al. (14). All other matebated at 30°C for 30 min and subsequently rials used were purchased as reagent grade. cooled below 20°C in an ice bath before measuring the absorbance at 475 nm against Methods a reagent blank containing the correspondReaction of p-nitrophenylglyoxal with ing amounts of (C) and (D). The arginine arginine. The effects of sodium ascorbate content of the sample was then determined
This paper describes a new method for the calorimetric determination of arginine residues in proteins by the use of p-nitrophenylglyoxal. The procedure requires no critical handling of reagents and is performed under mild conditions. There is no need to remove excess reagent before measuring the color produced because the absorption maximum of the product is greatly different from that of the reagent.
S Throughout this paper the imidazole ring of histidine and its derivatives is numbered according to IUPAC nomenclature.
4 p-Nitrophenylglyoxal should be stored desiccated in the dark at O-ST. Samples stored under these conditions were found to be stable for at least 18 months.
222
YAMASAKI,
SHIMER,
by comparison with a standard curve prepared using a stock solution of arginine of known concentration. When preparing a standard curve using free arginine or when assaying for arginine residues in intact proteins, the additions of (C) and (D) and the incubation step at 37°C for 3 h were omitted. All spectrophotometric measurements were performed using a Beckman Model 35 recording spectrophotometer. Protein concentrations were determined by measurement of their absorbance at 280 nm in deionized water or 10 mM sodium phosphate buffer, pH 7.0, using the following absorption coefficients (E:&): chicken ovotransferrin, 11.6 (15); bovine serum albumin, 6.60 (16); bovine pancreatic ribonuclease A, 6.95 (17); chicken ovalbumin, 7.50 (15); rabbit muscle creatine phosphokinase, 8.76 ( 18); yeast alcohol dehydrogenase, 12.6 (19). Concentrations of penguin ovalbumin and diaphorase solutions were determined by dry weight. Concentrations of arginyl-antifreeze glycopeptide 8 solutions were determined by amino acid analysis. Complete acid hydrolysis of protein samples was achieved with 6 M HCl at 110°C for 22 h in evacuated sealed tubes. Hydrolysates were analyzed on a Technicon auto analyzer. RESULTS Reaction of p-Nitrophenylglyoxal with Arginine
p-Nitrophenylglyoxal in 0.1 M sodium pyrophosphate, pH 9.0, was found to absorb maximally at 265 nm with a molar absorption coefficient of 8.1 x 103 M-l cm-l. Reaction of p-nitrophenylglyoxal with arginine at mildly alkaline pH resulted in the formation of a colored product whose absorbance maximum was found at 450 nm (Fig. 1). Constant purging of nitrogen gas into the reaction mixture allowed the orangered color to develop and remain. Shaking the colored reaction mixture in the presence of ordinary air caused a rapid fading of the
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I.., ‘......, %. ‘.. \
400
500 WAVELENGTH
600 (nm)
FIG. 1. Absorption spectra of the arginine-p-nitrophenylglyoxal reaction mixture. To 3.00 ml of 0.25 mM t-arginine in 0.1 M sodium pyrophosphate/O. 15 M sodium ascorbate, pH 9.0, was added 25 pi of 10% p-nitrophenylglyoxal in methanol (4.7 mM p-nitrophenylglyoxal in the reaction mixture). The absorbance scans were recorded after 70 min at 23°C. The concentration of p-nitrophenylglyoxal was approximately 20 times the concentration of arginine. Reaction mixture vs water (. .), reagent blank vs water (-), reaction mixture vs reagent blank (-).
color. Sodium ascorbate, at concentrations greater than 0.10 M, stabilized the color enough to permit the application of this reaction. Figure 2 shows the effect of sodium ascorbate concentration on color development. A concentration of 0.15 M sodium ascorbate was regarded as the best concentration when the color development in the blank was taken into account. The absorbance was routinely measured at 475 nm because of the high blank encountered at 450 nm (Fig. 1) in the presence of a 20-fold molar excess ofp-nitrophenylglyoxal. Ordinary room light appeared to have no effect on the color development. Hydroquinone, sodium sulfite, sodium borohydride, 3,5dihydroxybenzoic acid, sodium hydrosulfite, thiodiglycol, and sodium arsenite were ineffective in stabilizing the color. In general, the rate of color development increa‘sed with pH, p-nitrophenylglyoxal concentration, and temperature. The color
ARGININE
cl REACTION
RESIDUE DETERMINATION
50 TIME (min)
100
FIG. 2. Effect of sodium ascorbate concentration on color development. To 3.00 ml of 0.25 mM L-arginine in 0.1 M sodium pyrophosphate, pH 9.0, containing sodium ascorbate (concentration given below), was added 25 ~1 of lO%p-nitrophenylglyoxal in methanol. The reaction was allowed to proceed at 23°C. The concentrations are: 0.05 M sodium ascorbate (A), 0.10 M sodium ascorbate (0), 0.15 M sodium ascorbate (A), 0.20M sodium ascorbate (0).
appeared unstable at pH 9.5 and above, even in the presence of 0.15 M sodium ascorbate. At pH 10.0 and above, the blank, as well as the test solution, became turbid. For routine work pH 9.0 was therefore regarded as optimal. Aliquots of reagent (B) (10% methanolic g-nitrophenylglyoxal) larger than 25 ,ul led to turbidity in reaction volumes of 3.00 ml or less. For 3.00-ml reaction volumes, 25 ~1 of (B) gave the highest rate of color development without leading to turbidity. At 23°C color development was complete after 70 min and remained stable for an additional 30 min. By increasing the temperature to 3O”C, the reaction time was shortened to 30 min, but the color slowly faded about 10 min after reaching complete color development. Cooling (in an ice bath) below 20°C after achieving complete color development prevented this problem. Higher temperatures were unsuitable as the color was found too unstable to be of any value. Other compounds were tested for possible interfering absorbances that might occur as
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BY p-NITROPHENYLGLYOXAL
a result of side reactions. All 20 common amino acids found in proteins, as well as arginine methyl ester, cystine, cysteic acid, methionine sulfone, methionine sulfoxide, 2-mercaptoethanol, glutathione (reduced), sulfosalicylic acid, cystine dimethyl ester, dithiodiglycolic acid, urea, guanidine, sodium dodecyl sulfate, imidazole, l-methylhistidine, 3-methylhistidine, and the N”acetyl derivatives of arginine and histidine were individually tested at the same concentration (0.25 mM). Possible interfering compounds and pertinent related ones are shown in Table 1. All others tested but not listed in Table 1 gave absorbances of less than 0.005 at 475 nm. Free histidine reacted to give an absorbance -24% of that for arginine. It appeared that the free a-amino group must be present for this side reaction to occur since neither N”-acetylhistidine nor imidazole reacted to give an interfering absorbance. The imidazole ring must also play a role since 1-methylhistidine interfered TABLE ABSORPTION WITH
OF VARIOUS
1 COMPOUNDS
TREATED
p-NITROPHENYLGLYOXAL
Compound Arginine N”-Acetylarginine Arginine methyl ester Histidine N”Acetylhistidine Imidazole I-Methylhistidine 3-Methylhistidine N”-Acetyl-l-methylhistidine N”-Acetyl-3-methylhistidine Cysteine Glutathione (reduced) 2-Mercaptoethanol
Absorbance at 475 nm” 0.715 0.700 0.720 0.168 0 0 0.235 0 0.002 0 0.020 0.019 0.026
‘I To 3.00 ml of a 0.25 mM solution of the test compound in 0.1 M sodium pyrophosphate/O.lS M sodium ascorbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. After incubating for 30 min at 30°C. the solutions were cooled in an ice bath below 20°C and the absorbances at 475 nm were read against a reagent blank in I .O-cm cuvettes.
YAMASAKI,
0
0.2
ARGININE
CONCENTRATION
SHIMER,
0.4
(mM)
FIG. 3. Standard curve for arginine. To 3.00 ml of 0.03-0.33 mM L-arginine in 0.1 M sodium pyrophosphate/0.15 M sodium ascorbate, pH 9.0, was added 25 ~1 of 10% p-nitrophenylglyoxal in methanol. The reaction was carried out at 30°C for 30 min before reading the absorption at 475 nm against a reagent blank.
whereas 3-methylhistidine did not. Interference by free histidine and possibly amino terminal histidine in peptides may be prevented by acetylating the free amino group with acetic anhydride. At pH 8 complete acetylation of alanine is accomplished within 5 min with a twofold excess of acetic anhydride (20). Complete acetylation of histidine was obtained in less than 1 min at pH 9.0. Acetylation of 1-methylhistidine in this manner prevented the reaction which led to an interfering absorbance. Free sulfhydryl groups reacted with p-nitrophenylglyoxal, but the resulting absorbance at 475 nm was less than 3% of the absorbance yielded by arginine . Determination of Arginine Residues in Proteins
The methods described under Experimental Procedures were used to determine the number of arginine residues in several proteins of known amino acid composition. Beer’s law was obeyed in the range 0.030.33 mM arginine as shown by the standard curve in Fig. 3. Where intact proteins were
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assayed, the time for complete color development (to account for all of the arginine residues) varied with the protein. The time for complete color development with intact proteins was, in general, much longer than that with free arginine. For a more rapid quantitation of total arginine residues, best results were obtained when protein samples were predigested with a mixture of trypsin and subtilisin. It was found that 3-h digestion time was sufficient for the proteins studied. Table 2 compares the results of the total arginine determination of several proteins (via treatment with trypsin and subtilisin) with the corresponding known arginine contents. Samples treated with trypsin alone gave results intermediate of the results given by the two methods above. Arginine modified with p-nitrophenylglyoxal was found unstable to the conditions for the acid hydrolysis of proteins (-22% regeneration of arginine) and, therefore, the extent of modification as measured by spectrophotometry and amino acid analysis could not be compared. TABLE
2
ESTIMATIONOFTHENUMBEROFARGININERESIDUES PER MOLE OF PROTEIN No. of arginine
residues/ mol of protein”
p-Nitrophenylglyoxal method”
Protein Chicken ovotransferrin Bovine serum albumin Ribonuclease A Chicken ovalbumin Penguin ovalbumin Diaphorase Creatine phosphokinase Yeast alcohol dehydrogenase Arginylantifreeze glycopepfide
8
32.6 24.6 3.9 15.0 7.3 6.5 35.7 34.6 1.W
Expected’ 33.0 24.0 4.0 15.7’ 7.IP 6.6 35.1 31.7 1.0,
” Estimated on the basis of the following molecular weights: chicken ovotransfenin, 76,600 (21); bovine serum albumin, 67.OOil (22); ribonuclease A, 13.683 (23): chicken ovalbumin. 46.000 (15): penguin ovalbumin. 48.000 (24); diaphorase. 24,000 125); creatine phosphokinase. 82.600 (26): yeast alcohol dehydrogenase, 150,ooO (27): argmylantifreeze glycopeptide 8, 2800 t 10,281. 0 Protein samples were digested with a mixture of trypsin and subtilisin prior to assaying for nrginines (see Experimental Procedures). ’ On the basis of the known sequence and/or composition (see above references). n Obtained using the intact glycopeptide. * Obtained by amino acid analysis in our laboratory.
ARGININE
RESIDUE
DETERMINATION
Heat denaturation, alkali treatment, reduction and alkylation of disulfide bonds, and performic acid treatment were used in an attempt to disrupt the native structure of the protein and to fully expose all arginine residues to the reagent. These methods failed to give suitable results due to frequent precipitation of the treated samples. High concentrations of urea (4-8 M) were found unsuitable as they formed colorless solutions with p-nitrophenylglyoxal. Denaturation by 1% sodium dodecyl sulfate led to rates of color formation lower than those with intact proteins. Borate and 2-amino2-(hydroxymethyl)1,3-propanediol buffers could not be used as they severely reduced color formation. Reactions of phenylglyoxal (a homolog of p-nitrophenylglyoxal) with borate (29) and with 2-amino-2-(hydroxymethyl)-1,3-propanediol (30) have been reported. DISCUSSION
p-Nitrophenylglyoxal, in the presence of 0.15 M sodium ascorbate, can be used to reliably determine arginine residues in proteins. Since not all of the arginine residues in intact proteins are necessarily reactive, a method for disrupting the native structure was required for the quantitation of total arginine residues. Treatment with a mixture of trypsin and subtilisin gave satisfactory results in the examples studied. Other methods such as heat denaturation could be suitable for some proteins that do not precipitate under treatment. Workers have reported on modified Sakaguchi procedures that may be applicable to unhydrolyzed proteins, but anomalous results have been reported in certain cases (31). Izumi (32) has concluded that the Sakaguchi method is not applicable to the estimation of arginine residues in proteins without prior hydrolysis. Histidine also reacts with p-nitrophenylglyoxal to give a small interfering absorbance, but only if the free a-amino group is present. A possible explanation involves an attack on the imidazole ring by the pro-
BY p-NITROPHENYLGLYOXAL
225
tonated imine formed from the reaction of the a-amino group with the aldehyde group of p-nitrophenylglyoxal. A similar reaction with formaldehyde has been reported (33, 34). In the case of unhydrolyzed proteins, this interference does not usually arise since the a-amino groups of histidine are blocked in the form of peptide linkages. Only an N-terminal histidine residue, if available for reaction, would have the potential for interfering, and even then its contribution to the total absorbance would normally be small. Acetylation should usually be unnecessary when assaying unhydrolyzed proteins. The same argument can be applied to samples digested with trypsin and subtilisin except now there is a variety of N-terminal residues to deal with. However, it appears improbable that an amount of N-terminalhistidine-containing peptides sufficient to cause significant interference would be created. The data in this study seem to support this argument. Only one example in this study, yeast alcohol dehydrogenase, gave a high value for the total number of arginine residues. The procedures presented in this paper for the determination of arginine residues in proteins by p-nitrophenylglyoxal should easily be adopted for automation. One advantage p-nitrophenylglyoxal has over nearly all existing arginine color reagents is the mild conditions used for color development. Many proteins effectively retain their native structure at pH 9.0.p-Nitrophenylglyoxal might possibly be used to obtain information on the relative reactivities of the arginine residues in native proteins directly. It should be noted that a residue located at the surface of a protein or exposed to solvent may still appear unreactive (35,36). When determining arginine in an amino acid pool such as blood serum, where the amount of free histidine is likely to be significant, acetylation must be included.5 This 5 This is done rapidly and easily by acetylating with acetic anhydride (20). Preliminary results using rat blood serum showed 0.27 mM free arginine by p-nitro-
226
YAMASAKI,
SHIMER,
step is quick and simple and should be included in an automated program. It is interesting that upon reaction with arginine, p-nitrophenylglyoxal undergoes a large spectral shift, whereas phenylglyoxal,6 p-hydroxyphenylglyoxal (lo), and 4-hydroxy-3-nitrophenylglyoxal (9) do not. The colored p-nitrophenylglyoxal product has been used for direct determinations of arginines, while the stablep-hydroxyphenylglyoxal product (10) was used for preparations of chemically modified arginines. ACKNOWLEDGMENTS The authors thank David Sherman for preparing p-nitrophenylglyoxal, David T. Osuga for his many helpful discussions, Chris Howland for editorial assistance, and Clara Robison for typing the manuscript.
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phenylglyoxal while amino acid analysis showed 0.29 mM free arginine after removing serum proteins with sulfosalicylic acid. 6 Phenylglyoxal was found to absorb maximally at 252 nm in water. Phenylglyoxal-modified arginine was reported by Takahashi (37) to absorb maximally at 250 nm in water.
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FEENEY
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37. Takahashi, K. (1977) J. Biochem. (Tokyo) 81,403414.