- Email: [email protected]
Quantitative determination of monosubstituted guanidines: A comparative study of different procedures
Journal of Biochemical and Biophysical Methods, 7 (1983) 267-276 Elsevier
267
Quantitative determination of monosubstituted guanidines: A comparative study of different procedures Michael A. Parniak*, Gerald Lange and Thammaiah Viswanatha Department of Chemistry, Guelph - Waterloo Centre for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
(Received 15 December 1982) (Accepted 26 January 1983)
Summa~ Analysis of N2-acyl arginine derivatives as well as of arginine analogs lacking in a-amino function by Weber's modification of the Sakaguchi procedure yielded colored complexes with absorbance values approximately twice that obtained with an equivalent concentration of unmodified arginine. The limitations concerning the applicability of the various modifications of the Sakaguchi procedure as well as of the fluorimetric assay to the quantitative estimation of a variety of monosubstituted guanidines and proteins are discussed. Key words:i arginine; monosubstituted guanidine; Sakaguchi procedure; protein.
Introduction The Sakaguchi procedure has been used in this laboratory for the quantitative colorimetric determination of the arginine content of native and chemically modified proteins [1,2]. The procedure generally used is Weber's modification [3] of the original Sakaguchi procedure [4]. This method has been found to yield reliable and reproducible values for the arginine content of most proteins. In some cases, however, anomalies are evident. For example, Sakaguchi determinations of intact chymotrypsin indicate the presence of four arginine residues, whereas only three such residues are detectable by the automated amino acid analysis of the HCI hydrolysate of the protein [1,5]. Recent studies have shown that the hexapeptide Ala-Asn-Thr-Pro-Asp-Arg, corresponding to the segment 149-154 of the sequence of chymotrypsin [6], exhibits similar anomalous behaviour in Sakaguchi determina* Present address: Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
268 tions of arginine [7]. Thus, analysis of the intact peptide by the Sakaguchi procedure yields 2 mol of arginine while such determinations on its HC1 hydrolysates indicate the presence of 1 mol of the amino acid, the latter results being consistent with the data obtained by amino acid analysis. This discrepancy between the results of the analysis of the intact peptide and its acid hydrolysate points to the possibility of the response by a peptide bound arginine in Sakaguchi determinations being influenced by functional groups proximal to the amino acid. The arginine residue in the hexapeptide, besides being located at the C-terminus, is preceded by an aspartic acid residue. The reaction of N2-substituted arginines (compounds with structural features similar to that of arginine in the hexapeptide) and of other monosubstituted guanidines, with the Sakaguchi reagents forms the basis of this report. The work is extended to include a comparison of various modifications of the Sakaguchi procedure for the quantitative determination of monosubstituted guanidines. The utility of these modifications of the Sakaguchi procedure in the determination of the arginine content of unhydrolysed proteins is also presented.
Materials
1-Naphthol and 2,4-dichloro-l-naphthol were obtained from Eastman Kodak. 9,10-Phenanthrenequinone and bromine were from Aldrich. Sodium hypochlorite was from Fisher Scientific. NZ-Succinylarginine was prepared by standard procedures using arginine and succinic anhydride. The product, which was ninhydrin negative but gave a positive Sakaguchi reaction, could not be crystallized. The material was chromatographically pure as determined by thin-layer chromatography in several solvent systems, and gave a 1 : 1 ratio of arginine and succinic acid upon acid hydrolysis. N2-Acetylarginine was prepared as earlier described [8]. Arginine amide was prepared according to Ref. 9. All other monosubstituted guanidine derivatives were obtained from Sigma Chemical Co. Stock solutions of the guanidine derivatives in water were prepared gravimetrically. The concentration of stock solutions of NZ-succinylarginine was determined by automated amino acid analysis after acid hydrolysis. All proteins were obtained from Worthington Biochemical Corporation. Stock solutions of these proteins were prepared in 1 mM HC1. These solutions were stored at 4°C and used within 3 weeks of preparation. Glass-distilled deionized water was used throughout these studies.
Methods
Automated amino acid analysis was performed according to the procedure of Spackman et al. [10] using a Beckman model 120 B amino acid analyzer. Acid hydrolysis of proteins and other samples was carried out for 24 h at 105°C with 6 M HC1 in sealed, evacuated tubes.
269
Analytical procedures Unless otherwise specified, all procedures were carried out at room temperature. All standard curves were constructed using known concentrations of arginine.
L Method of Weber [3] The reagents used are: (A) 10% KOH. (B) 0.1% 1-naphthol in 50% ethanol. (C) 5% urea. (D) 0.64 ml bromine in 100 ml of 5% KOH. This reagent is prepared fresh daily. 1 ml of the sample solution (containing 0.1-0.5/~mol of monosubstituted guanidine) is mixed with 1 ml of A and 1 ml of B and allowed to stand for 3 min. Then 1 ml of C is added and the solution is again mixed, followed by the addition of 2 ml of D with continuous shaking. The solution is allowed to stand for 20 min, then the absorbance at 520 nm is determined against a suitable reagent blank. II. Method of Van Pilsurn et al. [11] The reagents used are: (A) 10% N a O H containing 20 m g / m l of thymine. (B) 0.04% l-naphthol in absolute ethanol. (C) 1% sodium hypochlorite (prepared fresh daily). (D) 2% sodium thiosulfate. 1 ml of the sample solution (containing 0.05-0.25 /Lmol of monosubstituted guanidine) is cooled on ice, then 0.5 ml of a 1:1 mixture of reagents A and B, prepared fresh daily, is added. After mixing, 0.2 ml of C is added with continuous shaking. Exactly 1 min later, 0.2 ml of D is added and the solution is vigorously shaken. The absorbance of the sample at 515 nm is determined against a suitable reagent blank. The color is stable in the cold for several hours. If the samples are allowed to warm to room temperature, the color fades rapidly and disappears within 10 min. lI1. Method of Pesez and Bartos [12] The reagents used are: (A) 1% thymine in 1 M NaOH. (B) 2% 8-hydroxyquinoline in absolute ethanol. (C) Dilute aqueous sodium hypochlorite (0.316 g active chlorine in 100 ml of water). 1 ml of the sample solution (containing 0.05-0.25 /~mol of monosubstituted guanidine) is mixed with 0.5 ml of A and 0.1 ml of B. The solution is cooled on ice for 2 min, and 0.5 ml of precooled C is added with mixing. The solution is allowed to stand for 3 min on ice, and 3 ml of methanol is added. The absorbance at 490 nm is determined against a suitable reagent blank. The color formed is stable for approximately 2 h.
270
IV. Method of Messineo [13] The reagents used are as follows: (A) 300 mg of potassium iodide in 100 ml of water. (B) 2 g of potassium sodium tartrate in 100 ml of 5 M KOH. To this solution is added 100 mg of 2,4-dichloro-l-naphthol and 180 ml of absolute ethanol. Approximately 3 ml of commercial 4-6% sodium hypochlorite is added, and the solution is allowed to stand for at least 1 h prior to use. For each new batch of sodium hypochlorite, the exact volume to be used is determined by adding various amounts of hypochlorite to the tartrate/naphthol solution and measuring the absorbance at 400 nm after 30 min against a suitable reagent blank. The amount of sodium hypochlorite which gives the maximum absorbance at 400 nm is the optimum amount to be used in the preparation of reagent B. This reagent is stable for several weeks when stored at 4°C. (C) Approximately 5 ml of commercial 4-6% sodium hypochlorite diluted to 100 ml with water. The optimum concentration of hypochlorite required is determined as described for reagent B. Reagent C is prepared fresh daily. 1 ml of sample (containing 0. I-0.5/.tmol of monosubstituted guanidine) is mixed with 1 ml of A and 3 ml of B, and the solution is allowed to stand for 1 h. After the addition of 1 ml of C, the solution is mixed and allowed to stand for 10 min. The absorbance at 520 nm is determined against a suitable reagent blank.
V. Phenanthrenequinone procedure [12] The reagents used are: (A) 0.05% phenanthrenequinone in absolute ethanol. The phenanthrenequinone is recrystaUized several times from ethanol prior to use. (B) 10 M NaOH. To 0.5 ml of sample (containing 0.01-0.1 /~mol monosubstituted guanidine) is added 0.5 ml of A and 0.1 ml of B. After mixing, the solution is allowed to stand for 30 min. Then 0.25 ml of concentrated HC1 and 2.5 ml of water are added with mixing. The fluorescence of the sample is determined using excitation and emission wavelengths of 360 and 400 nm, respectively, with appropriate reagent blank serving as control.
Results
The arginine content of solutions of N2-succinylarginine and N2-acetylarginine was determined by Weber's modification of the Sakaguchi procedure [3]. The values thus obtained were compared with those given both by amino acid analysis and by Sakaguchi determinations of identical aliquots after acid hydrolysis. The linear relationship between the absorbance given in the Sakaguchi procedure and the arginine content of the samples as determined by amino acid analysis is shown in Fig. 1. The slopes of the lines indicate that the color yield of the unhydrolysed samples was approximately twice that of identical samples after acid hydrolysis. In Weber's procedure for the determination of monosubstituted guanidines, the
271
1.5
o 0,6
¢N L£)
¢N Lt'3
1.0
z z <
~
n"
o (,,,3 t.n <
0.4
t.n n-.
0.5
o U')
/ t l , / "/9"¢
rn
0.2
//4 I
0.2
L
t
0.4
CONCENTRATION (#mol)
L 6
I 12
L 18
2t4
310
TIME AFTER ADDITION OF HYPOBROMITE (min)
Fig. 1. Absorbance at 520 nm as a function of the concentration of arginine derivative. Various amounts of N2-succinylarginine (e) or N2-acetylarginine (©) were subjected to Sakaguchi analysis using procedure I as described in Methods. The upper line ( ) corresponds to samples prior to acid hydrolysis, whereas the lower line (. . . . . . ) corresponds to identical aliquots subsequent to acid hydrolysis. Fig. 2. Decay of the colored product formed upon reaction of Sakaguchi reagents with monosubstituted guanidines. Sakaguchi procedure I as described in Methods was used. Arginine (©), N2-succinylarginine (zx), and N2-acetylarginine (e) were each 0.2 gmol in a total volume of 5 ml. Absorbance measurements were started 1 rain after the addition of hypobromite in order to circumvent possible interference due to the evolution of gas normally seen upon mixing the reagents.
a b s o r b a n c e of the colored c o m p l e x undergoes an initial r a p i d decay. Consequently, an essential r e q u i r e m e n t for achieving r e p r o d u c i b l e results is that the a b s o r b a n c e values of s a m p l e s be r e c o r d e d 20 min after the a d d i t i o n of h y p o b r o m i t e reagent. A s shown in Fig. 2, the N2-acylarginines differ from u n m o d i f i e d arginine in b o t h the m a g n i t u d e of the d e c a y in a b s o r b a n c e after the a d d i t i o n of h y p o b r o m i t e as well as in the stability of the colored complex. N o further decrease in a b s o r b a n c e at 520 n m was n o t e d 10 m i n after the a d d i t i o n of h y p o b r o m i t e to solutions of the a c y l a t e d c o m p o u n d s . However, a c o n t i n u a l decrease in a b s o r b a n c e was seen with solutions of arginine even 20 m i n after the a d d i t i o n of h y p o b r o m i t e . A wide variety of m o n o s u b s t i t u t e d g u a n i d i n e c o m p o u n d s , in a d d i t i o n to N2-succ i n y l a r g i n i n e a n d N2-acetylarginine, give increased a b s o r p t i v i t y relative to arginine w h e n m e a s u r e d with W e b e r ' s m o d i f i c a t i o n of the Sakaguchi p r o c e d u r e (Table 1, m e t h o d I). A b s o r b a n c e was linear with respect to c o n c e n t r a t i o n for all the comp o u n d s studied. T h e a b s o r p t i o n s p e c t r u m of the c o l o r e d p r o d u c t o b t a i n e d with arginine shows a b r o a d m a x i m u m b e t w e e n 490 a n d 520 nm. T h e c o m p o u n d s which
272 TABLE 1 Q U A N T I T A T I V E D E T E R M I N A T I O N OF M O N O S U B S T I T U T E D G U A N I D I N E S BY V A R I O U S M O D I F I C A T I O N S OF T H E S A K A G U C H I P R O C E D U R E All values are relative to the color yield or relative fluorescence given by equimolar amounts of a standard solution of arginine. All values in the table are means _+S.D. for at least six determinations over a range of concentrations. N.D., not determined. Procedure: I, Weber [3]; II, Van Pilsum et al. [l 1]; Ill, Pesez and Bartos [12]; IV, Messineo [13]; V, fluorimetric method of Perez and Bartos [12]. Compound
N2-Acetylarginine N 2-Succinylarginine Argininic acid Guanidoacetic acid Guanidopropionic acid Guanidobutyric acid Guanidovaleric acid Guanidocaproic acid Guanidosuccinic acid Agmatine Acetylagmatine Arginine amide N Z-Acetylarginine amide
Procedure I
II
IlI
IV
V
1.86_+0.11 1.74+0.10 1.61 +0.16 0.95+-0.07 1.83+0. I0 1.83+-0.12 1.74+-0.08 1.76+-0.15 0.41 + 0.06 1.29_+0.10 1.51 +- 0.03 1.23+-0.07 1.36 +- 0.06
1.12___0.05 1.18+0.08 1.03+0.05 1.13+-0.06 1.17+0.05 1.19+-0.04 1.17+-0.04 1.13+-0.06 0.43 + 0.04 0.97+0.06 0.96 + 0.03 0.85+-0.04 0.95 +- 0.04
1.13_+0.04 0.92+-0.06 N.D. 0,87+-0.06 1.08+0.08 1.03+-0.07 0.94+-0.08 0.89+0.04 N.D. 1.08+-0.03 N.D. 0.75_+0.05 N.D.
1.31_+0.22 1.12_+0.20 N.D. 0.87+0.14 1.26+0.26 1.26+-0.18 1.39+-0.15 1.48+-0.14 N.D. 1.03+-0.08 N.D. 0.97_+0.09 N.D.
0.95+_0.11 1.00+0.01 1.14+-0.10 0.99+-0.44 0.99+-0.05 1.09+-0.03 1.08___0.04 1.11___0.07 0.65 +- 0.03 1.06+-0.17 1.11 _ 0.09 0.92+-0.13 0.80 ± 0.08
give enhanced color yields relative to arginine exhibit relatively sharp absorbance maxima at 530 nm (Fig. 3). Since Weber's modification of the Sakaguchi procedure gives widely varying color yields with the various monosubstituted guanidines studied, the utility of other modifications of the Sakaguchi procedure was investigated. The methods studied were those described by Van Pilsum et al. [11], Messineo [13] and Pesez and Bartos [12]. In addition, a well documented fluorimetric procedure, the reaction of phenanthrenequinone with monosubstituted guanidines [12,14,15] was employed. The results of these experiments are summarized in Table 1. In all cases, a linear relationship between absorbance (or fluorescence intensity) and the concentration of monosubstituted guanidine was observed. The results obtained by the procedures of Van Pilsum et al. [11] and Pesez and Bartos [12] and by the fluorimetric method [12] show reasonable correspondence (_+ 10%) to the actual concentrations of monosubstituted guanidines employed in the experiment. Such is not the case in the determinations involving Weber's [3] and Messineo's [13] modifications of the Sakaguchi procedure. The applicability of the various Sakaguchi methods to the determination of the arginine content of intact proteins was studied. Table 2 compares the arginine content of the protein solutions obtained with the various colorimetric and fluorimetric procedures with values obtained by automated amino acid analysis of acid hydrolysates of these same protein solutions. A linear relationship between the concentration of protein and absorbance was noted with the procedures of Weber
273
0.8
0.6 Z
o ,~ 0.4
'\
/¢ t
¢ /
\
0.2
,
i
480
,
i
500
,
i
,
520
i
540
,
560
A (nm)
Fig. 3. Absorbance spectra of monosubstituted guanidines obtained using Sakaguchi procedure I. The samples arginine (. . . . . . ) and guanidobutyric acid ( ) were present at a concentration of 0.3 ktmol in the assay.
TABLE 2 DETERMINATION OF THE ARGININE CONTENT OF INTACT PROTEINS BY VARIOUS MODIFICATIONS OF THE SAKAGUCHI PROCEDURE Procedures I - V are as described in Methods. Automated amino acid analysis performed on aliquots after acid hydrolysis (6 M HCI, 24 h, in vacuo) according to the procedure described in Ref. 10. The expected number of arginine residues per molecule is based on the reported amino acid compositions of the proteins as described in the references provided in the table. Protein
Residues per molecule I
II
III
IV
V
Amino acid Expected Reference analysis
~bonuclease 4.2±0.1 1 . 2 ± 0 . 1 0 . 9 ± 0 . 1 1.9±0.1 6.1±0.1 3.9 Lysozyme 10.3±0.5 7.3±0.1 6.2±0.2 9.4± 1.1 7.7±0.5 9.9 a-Chymot~psin 4.3±0.3 2.4±0.1 1.6±0.1 2.8±0.3 3.6±0.1 2.8 ChymotrypsinogenA 5.0±0.2 2.8±0.1 1.4±0.1 3.4±0.2 3.4±0.1 3.6 Trypsin 2.6±0.2 1.3±0.1 1.0±0.1 1.6±0.2 3.5±0.1 1.9 SubtiHsin 3.7±0.3 1.6±0.1 1.3±0.1 2.0±0.2 4.3±0.1 1.9 Thyrotropin 3.6±0.2 2.1±0.1 1.9±0.1 3.0±0.5 5.4±0.1 3.6
4.0 11.0 3.0 4.0 2.0 2.0 4.0
18 19 6 6 20 21 21
274 [3], Van Pilsum et al. [1 1], and Pesez and Bartos [12]. The method of Messineo [13] and the fluorimetric phenanthrenequinone procedure [12] failed to yield such a relationship.
Discussion
N2-Succinylarginine was selected to serve as a model to elucidate the unusual reactivity of an arginine residue located in the hexapeptide segment corresponding to the sequence 149-154 of chymotrypsin, towards the Sakaguchi reaction. This arginine gives approximately twice the color yield as compared to unmodified arginine in Weber's modification [3] of the Sakaguchi procedure [4]. N 2Acetylarginine, upon reaction with these same reagents, also gave nearly twice the amount of colored product as that obtained with an equivalent amount of arginine. Subsequent work indicated that a wide variety of monosubstituted guanidines exhibited this feature. Compounds similar in structure to arginine, but for acylation or elimination of the c~-amino group, gave increased absorptivity upon reaction with the Sakaguchi reagents as described by Weber [3]. The enhanced color given by N2-acylarginine derivatives appears to be due, in part, to stabilization of the colored complex formed upon reaction of the guanidine derivative with the Sakaguchi reagents. In addition, the colored product produced by the acylated compounds possesses different spectral characteristics than the corresponding arginine chromophore. Arginine derivatives showing enhanced color with Weber's reagents failed to exhibit significant enhancement when tested by several other modifications of the Sakaguchi procedure. This may indicate that the enhanced color noted with the Weber method is an inherent anomaly of that procedure. Thus, the standard curves prepared with arginine cannot be used to determine the concentrations of solutions of other monosubstituted guanidines when Weber's modification [3] of the Sakaguchi procedure is employed. Several reports have suggested that the Sakaguchi procedure may be applicable to the estimation of arginine in intact proteins [1,13], whereas others have stated that the method is unsuitable [16]. The current investigations tend to support this latter opinion. Surprisingly, Weber's modification [3] of the Sakaguchi procedure proved to be the most accurate, when tested with proteins such as ribonuclease and lysozyme. Serine proteases, however, indicate the presence of an additional Sakaguchi-positive component, in agreement with previous findings [1,5]. The demonstration, by this procedure, of an additional Sakaguchi positive component in the hexapeptide fragment 149-154 of chymotrypsin is consistent with similar observations recorded with N2-acyl arginine derivatives. The C-terminal location of the arginine in the hexapeptide does not appear to be responsible for the anomaly in view of the manifestation of the phenomenon in intact chymotrypsin. Furthermore, neither of the two arginine residues in trypsin, a serine protease which has also been shown to possess an additional Sakaguchi-positive component [1], is preceded by an aspartic acid residue. Hence, it is difficult to attribute the additional
275 S a k a g u c h i - p o s i t i v e c o m p o n e n t in c h y m o t r y p s i n to the A s p - A r g sequence p r e s e n t in the h e x a p e p t i d e segment. Thus, the a d d i t i o n a l Sakaguchi c o m p o n e n t a p p a r e n t with W e b e r ' s p r o c e d u r e a p p e a r s to be an inherent feature of serine proteases for as yet u n k n o w n reasons. The other m o d i f i c a t i o n s of the Sakaguchi p r o c e d u r e tested d i d n o t yield reliable estimates of the arginine c o n t e n t of a n y of the p r o t e i n s e m p l o y e d in these studies. The fluorimetric p h e n a n t h r e n e q u i n o n e p r o c e d u r e used [12] also d i d not give reliable values for the arginine content of proteins. S m i t h a n d M a c Q u a r r i e [17] have recently shown that, with suitable modifications, this p r o c e d u r e can yield reliable estimates of the arginine c o n t e n t of intact proteins. However, o p t i m a l reaction c o n d i t i o n s m u s t be d e t e r m i n e d for each p r o t e i n tested. This d i s a d v a n t a g e is offset s o m e w h a t b y the reliability of the m e t h o d when c o m p a r e d with those a t t a i n a b l e in p r o c e d u r e s discussed in the present report. In addition, the fluorimetric p r o c e d u r e is c o n s i d e r a b l y m o r e sensitive than the Sakaguchi method.
Simplified description of the method and its applications The applicability of the various modifications of the Sakaguchi procedure for the determination of arginine residues in intact proteins was investigated. The results obtained were compared with those determined by the ion-exchange chromatography of the HC1 hydrolysates of proteins. These studies suggest that none of the modifications of the Sakaguchi procedure is suitable for the determination of the arginine content of proteins.
Acknowledgement T h e research was s u p p o r t e d b y the N a t u r a l Sciences a n d Engineering R e s e a r c h C o u n c i l of C a n a d a .
References 1 Tomlinson, G. and Viswanatha, T. (1974) Anal. Biochem. 60, 15-24 2 Martin, B.M., Fliss, H., Rama Murthy, J., Stepanik, T. and Viswanatha, T. (1975) Life Sci. 17, 1265-1268 3 Weber, C.J. (1930) J. Biol. Chem. 86, 217 4 Sakaguchi, S. (1925) J. Biochem. (Tokyo) 5, 25-31 5 Rickert, W.S. and Viswanatha, T. (1967) Biochem. Biophys. Res. Commun. 28, 1028-1033 6 Dixon, G.H. and Neurath, H. (1957) J. Biol. Chem. 225, 1040-1059 7 Fliss, H. (1977) Ph.D. Thesis, University of Waterloo; Stepanik, T.M. (1977) Ph.D. Thesis, University of Waterloo, Ontario 8 Greenstein, J.P. and Winitz, M. (1961) Chemistry of the Amino Acids, Vol. 3, p. 1850. Wiley, New York 9 Yang, P.S. and Rising, M.M. (1931) J. Am. Chem. Soc. 53, 3183-3184 10 Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 11 Van Pilsum, J.F., Martin, R.P., Kito, E. and Hess, J. (1956) J. Biol. Chem. 222, 225-236 12 Pesez, M. and Bartos, J. (1974) Colorimetric and Fluorometric Analysis of Organic Compounds and Drugs. Marcel Dekker, New York
276 13 14 15 16 17 18 19 20
Messineo, L. (1966) Arch. Biochem. Biophys. 117, 534-540 Yamada, S. and Itano, H.A. (1966) Biochim. Biophys. Acta 130, 538-540 Itano, H.A., Hiroto, K., Kawaski, I. and Yamada, S. (1976) Anal. Biochem. 76, 134-141 Izumi, Y. (1965) Anal. Biochem. 12, 1-7 Smith, R.E. and MacQuarrie, R. (1978) Anal. Biochem. 90, 246-255. Sherwood, L.M. and Potts, J.T. Jr. (1965) J. Biol. Chem. 240, 3799-3805 Steiner, R.F. (1964) Biochim. Biophys. Acta 79, 51-63 Walsh, K.A., Kauffman, D.L., Sampath Kumar, K.S.V. and Neurath, H. (1964) Proc. Natl. Acad, Sc U.S.A. 51,301-308 21 Croft, L.R. (1973) Handbook of Protein Sequences. Joynson-Bruvers, Oxford
267
Quantitative determination of monosubstituted guanidines: A comparative study of different procedures Michael A. Parniak*, Gerald Lange and Thammaiah Viswanatha Department of Chemistry, Guelph - Waterloo Centre for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
(Received 15 December 1982) (Accepted 26 January 1983)
Summa~ Analysis of N2-acyl arginine derivatives as well as of arginine analogs lacking in a-amino function by Weber's modification of the Sakaguchi procedure yielded colored complexes with absorbance values approximately twice that obtained with an equivalent concentration of unmodified arginine. The limitations concerning the applicability of the various modifications of the Sakaguchi procedure as well as of the fluorimetric assay to the quantitative estimation of a variety of monosubstituted guanidines and proteins are discussed. Key words:i arginine; monosubstituted guanidine; Sakaguchi procedure; protein.
Introduction The Sakaguchi procedure has been used in this laboratory for the quantitative colorimetric determination of the arginine content of native and chemically modified proteins [1,2]. The procedure generally used is Weber's modification [3] of the original Sakaguchi procedure [4]. This method has been found to yield reliable and reproducible values for the arginine content of most proteins. In some cases, however, anomalies are evident. For example, Sakaguchi determinations of intact chymotrypsin indicate the presence of four arginine residues, whereas only three such residues are detectable by the automated amino acid analysis of the HCI hydrolysate of the protein [1,5]. Recent studies have shown that the hexapeptide Ala-Asn-Thr-Pro-Asp-Arg, corresponding to the segment 149-154 of the sequence of chymotrypsin [6], exhibits similar anomalous behaviour in Sakaguchi determina* Present address: Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, MD 20205, U.S.A.
0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
268 tions of arginine [7]. Thus, analysis of the intact peptide by the Sakaguchi procedure yields 2 mol of arginine while such determinations on its HC1 hydrolysates indicate the presence of 1 mol of the amino acid, the latter results being consistent with the data obtained by amino acid analysis. This discrepancy between the results of the analysis of the intact peptide and its acid hydrolysate points to the possibility of the response by a peptide bound arginine in Sakaguchi determinations being influenced by functional groups proximal to the amino acid. The arginine residue in the hexapeptide, besides being located at the C-terminus, is preceded by an aspartic acid residue. The reaction of N2-substituted arginines (compounds with structural features similar to that of arginine in the hexapeptide) and of other monosubstituted guanidines, with the Sakaguchi reagents forms the basis of this report. The work is extended to include a comparison of various modifications of the Sakaguchi procedure for the quantitative determination of monosubstituted guanidines. The utility of these modifications of the Sakaguchi procedure in the determination of the arginine content of unhydrolysed proteins is also presented.
Materials
1-Naphthol and 2,4-dichloro-l-naphthol were obtained from Eastman Kodak. 9,10-Phenanthrenequinone and bromine were from Aldrich. Sodium hypochlorite was from Fisher Scientific. NZ-Succinylarginine was prepared by standard procedures using arginine and succinic anhydride. The product, which was ninhydrin negative but gave a positive Sakaguchi reaction, could not be crystallized. The material was chromatographically pure as determined by thin-layer chromatography in several solvent systems, and gave a 1 : 1 ratio of arginine and succinic acid upon acid hydrolysis. N2-Acetylarginine was prepared as earlier described [8]. Arginine amide was prepared according to Ref. 9. All other monosubstituted guanidine derivatives were obtained from Sigma Chemical Co. Stock solutions of the guanidine derivatives in water were prepared gravimetrically. The concentration of stock solutions of NZ-succinylarginine was determined by automated amino acid analysis after acid hydrolysis. All proteins were obtained from Worthington Biochemical Corporation. Stock solutions of these proteins were prepared in 1 mM HC1. These solutions were stored at 4°C and used within 3 weeks of preparation. Glass-distilled deionized water was used throughout these studies.
Methods
Automated amino acid analysis was performed according to the procedure of Spackman et al. [10] using a Beckman model 120 B amino acid analyzer. Acid hydrolysis of proteins and other samples was carried out for 24 h at 105°C with 6 M HC1 in sealed, evacuated tubes.
269
Analytical procedures Unless otherwise specified, all procedures were carried out at room temperature. All standard curves were constructed using known concentrations of arginine.
L Method of Weber [3] The reagents used are: (A) 10% KOH. (B) 0.1% 1-naphthol in 50% ethanol. (C) 5% urea. (D) 0.64 ml bromine in 100 ml of 5% KOH. This reagent is prepared fresh daily. 1 ml of the sample solution (containing 0.1-0.5/~mol of monosubstituted guanidine) is mixed with 1 ml of A and 1 ml of B and allowed to stand for 3 min. Then 1 ml of C is added and the solution is again mixed, followed by the addition of 2 ml of D with continuous shaking. The solution is allowed to stand for 20 min, then the absorbance at 520 nm is determined against a suitable reagent blank. II. Method of Van Pilsurn et al. [11] The reagents used are: (A) 10% N a O H containing 20 m g / m l of thymine. (B) 0.04% l-naphthol in absolute ethanol. (C) 1% sodium hypochlorite (prepared fresh daily). (D) 2% sodium thiosulfate. 1 ml of the sample solution (containing 0.05-0.25 /Lmol of monosubstituted guanidine) is cooled on ice, then 0.5 ml of a 1:1 mixture of reagents A and B, prepared fresh daily, is added. After mixing, 0.2 ml of C is added with continuous shaking. Exactly 1 min later, 0.2 ml of D is added and the solution is vigorously shaken. The absorbance of the sample at 515 nm is determined against a suitable reagent blank. The color is stable in the cold for several hours. If the samples are allowed to warm to room temperature, the color fades rapidly and disappears within 10 min. lI1. Method of Pesez and Bartos [12] The reagents used are: (A) 1% thymine in 1 M NaOH. (B) 2% 8-hydroxyquinoline in absolute ethanol. (C) Dilute aqueous sodium hypochlorite (0.316 g active chlorine in 100 ml of water). 1 ml of the sample solution (containing 0.05-0.25 /~mol of monosubstituted guanidine) is mixed with 0.5 ml of A and 0.1 ml of B. The solution is cooled on ice for 2 min, and 0.5 ml of precooled C is added with mixing. The solution is allowed to stand for 3 min on ice, and 3 ml of methanol is added. The absorbance at 490 nm is determined against a suitable reagent blank. The color formed is stable for approximately 2 h.
270
IV. Method of Messineo [13] The reagents used are as follows: (A) 300 mg of potassium iodide in 100 ml of water. (B) 2 g of potassium sodium tartrate in 100 ml of 5 M KOH. To this solution is added 100 mg of 2,4-dichloro-l-naphthol and 180 ml of absolute ethanol. Approximately 3 ml of commercial 4-6% sodium hypochlorite is added, and the solution is allowed to stand for at least 1 h prior to use. For each new batch of sodium hypochlorite, the exact volume to be used is determined by adding various amounts of hypochlorite to the tartrate/naphthol solution and measuring the absorbance at 400 nm after 30 min against a suitable reagent blank. The amount of sodium hypochlorite which gives the maximum absorbance at 400 nm is the optimum amount to be used in the preparation of reagent B. This reagent is stable for several weeks when stored at 4°C. (C) Approximately 5 ml of commercial 4-6% sodium hypochlorite diluted to 100 ml with water. The optimum concentration of hypochlorite required is determined as described for reagent B. Reagent C is prepared fresh daily. 1 ml of sample (containing 0. I-0.5/.tmol of monosubstituted guanidine) is mixed with 1 ml of A and 3 ml of B, and the solution is allowed to stand for 1 h. After the addition of 1 ml of C, the solution is mixed and allowed to stand for 10 min. The absorbance at 520 nm is determined against a suitable reagent blank.
V. Phenanthrenequinone procedure [12] The reagents used are: (A) 0.05% phenanthrenequinone in absolute ethanol. The phenanthrenequinone is recrystaUized several times from ethanol prior to use. (B) 10 M NaOH. To 0.5 ml of sample (containing 0.01-0.1 /~mol monosubstituted guanidine) is added 0.5 ml of A and 0.1 ml of B. After mixing, the solution is allowed to stand for 30 min. Then 0.25 ml of concentrated HC1 and 2.5 ml of water are added with mixing. The fluorescence of the sample is determined using excitation and emission wavelengths of 360 and 400 nm, respectively, with appropriate reagent blank serving as control.
Results
The arginine content of solutions of N2-succinylarginine and N2-acetylarginine was determined by Weber's modification of the Sakaguchi procedure [3]. The values thus obtained were compared with those given both by amino acid analysis and by Sakaguchi determinations of identical aliquots after acid hydrolysis. The linear relationship between the absorbance given in the Sakaguchi procedure and the arginine content of the samples as determined by amino acid analysis is shown in Fig. 1. The slopes of the lines indicate that the color yield of the unhydrolysed samples was approximately twice that of identical samples after acid hydrolysis. In Weber's procedure for the determination of monosubstituted guanidines, the
271
1.5
o 0,6
¢N L£)
¢N Lt'3
1.0
z z <
~
n"
o (,,,3 t.n <
0.4
t.n n-.
0.5
o U')
/ t l , / "/9"¢
rn
0.2
//4 I
0.2
L
t
0.4
CONCENTRATION (#mol)
L 6
I 12
L 18
2t4
310
TIME AFTER ADDITION OF HYPOBROMITE (min)
Fig. 1. Absorbance at 520 nm as a function of the concentration of arginine derivative. Various amounts of N2-succinylarginine (e) or N2-acetylarginine (©) were subjected to Sakaguchi analysis using procedure I as described in Methods. The upper line ( ) corresponds to samples prior to acid hydrolysis, whereas the lower line (. . . . . . ) corresponds to identical aliquots subsequent to acid hydrolysis. Fig. 2. Decay of the colored product formed upon reaction of Sakaguchi reagents with monosubstituted guanidines. Sakaguchi procedure I as described in Methods was used. Arginine (©), N2-succinylarginine (zx), and N2-acetylarginine (e) were each 0.2 gmol in a total volume of 5 ml. Absorbance measurements were started 1 rain after the addition of hypobromite in order to circumvent possible interference due to the evolution of gas normally seen upon mixing the reagents.
a b s o r b a n c e of the colored c o m p l e x undergoes an initial r a p i d decay. Consequently, an essential r e q u i r e m e n t for achieving r e p r o d u c i b l e results is that the a b s o r b a n c e values of s a m p l e s be r e c o r d e d 20 min after the a d d i t i o n of h y p o b r o m i t e reagent. A s shown in Fig. 2, the N2-acylarginines differ from u n m o d i f i e d arginine in b o t h the m a g n i t u d e of the d e c a y in a b s o r b a n c e after the a d d i t i o n of h y p o b r o m i t e as well as in the stability of the colored complex. N o further decrease in a b s o r b a n c e at 520 n m was n o t e d 10 m i n after the a d d i t i o n of h y p o b r o m i t e to solutions of the a c y l a t e d c o m p o u n d s . However, a c o n t i n u a l decrease in a b s o r b a n c e was seen with solutions of arginine even 20 m i n after the a d d i t i o n of h y p o b r o m i t e . A wide variety of m o n o s u b s t i t u t e d g u a n i d i n e c o m p o u n d s , in a d d i t i o n to N2-succ i n y l a r g i n i n e a n d N2-acetylarginine, give increased a b s o r p t i v i t y relative to arginine w h e n m e a s u r e d with W e b e r ' s m o d i f i c a t i o n of the Sakaguchi p r o c e d u r e (Table 1, m e t h o d I). A b s o r b a n c e was linear with respect to c o n c e n t r a t i o n for all the comp o u n d s studied. T h e a b s o r p t i o n s p e c t r u m of the c o l o r e d p r o d u c t o b t a i n e d with arginine shows a b r o a d m a x i m u m b e t w e e n 490 a n d 520 nm. T h e c o m p o u n d s which
272 TABLE 1 Q U A N T I T A T I V E D E T E R M I N A T I O N OF M O N O S U B S T I T U T E D G U A N I D I N E S BY V A R I O U S M O D I F I C A T I O N S OF T H E S A K A G U C H I P R O C E D U R E All values are relative to the color yield or relative fluorescence given by equimolar amounts of a standard solution of arginine. All values in the table are means _+S.D. for at least six determinations over a range of concentrations. N.D., not determined. Procedure: I, Weber [3]; II, Van Pilsum et al. [l 1]; Ill, Pesez and Bartos [12]; IV, Messineo [13]; V, fluorimetric method of Perez and Bartos [12]. Compound
N2-Acetylarginine N 2-Succinylarginine Argininic acid Guanidoacetic acid Guanidopropionic acid Guanidobutyric acid Guanidovaleric acid Guanidocaproic acid Guanidosuccinic acid Agmatine Acetylagmatine Arginine amide N Z-Acetylarginine amide
Procedure I
II
IlI
IV
V
1.86_+0.11 1.74+0.10 1.61 +0.16 0.95+-0.07 1.83+0. I0 1.83+-0.12 1.74+-0.08 1.76+-0.15 0.41 + 0.06 1.29_+0.10 1.51 +- 0.03 1.23+-0.07 1.36 +- 0.06
1.12___0.05 1.18+0.08 1.03+0.05 1.13+-0.06 1.17+0.05 1.19+-0.04 1.17+-0.04 1.13+-0.06 0.43 + 0.04 0.97+0.06 0.96 + 0.03 0.85+-0.04 0.95 +- 0.04
1.13_+0.04 0.92+-0.06 N.D. 0,87+-0.06 1.08+0.08 1.03+-0.07 0.94+-0.08 0.89+0.04 N.D. 1.08+-0.03 N.D. 0.75_+0.05 N.D.
1.31_+0.22 1.12_+0.20 N.D. 0.87+0.14 1.26+0.26 1.26+-0.18 1.39+-0.15 1.48+-0.14 N.D. 1.03+-0.08 N.D. 0.97_+0.09 N.D.
0.95+_0.11 1.00+0.01 1.14+-0.10 0.99+-0.44 0.99+-0.05 1.09+-0.03 1.08___0.04 1.11___0.07 0.65 +- 0.03 1.06+-0.17 1.11 _ 0.09 0.92+-0.13 0.80 ± 0.08
give enhanced color yields relative to arginine exhibit relatively sharp absorbance maxima at 530 nm (Fig. 3). Since Weber's modification of the Sakaguchi procedure gives widely varying color yields with the various monosubstituted guanidines studied, the utility of other modifications of the Sakaguchi procedure was investigated. The methods studied were those described by Van Pilsum et al. [11], Messineo [13] and Pesez and Bartos [12]. In addition, a well documented fluorimetric procedure, the reaction of phenanthrenequinone with monosubstituted guanidines [12,14,15] was employed. The results of these experiments are summarized in Table 1. In all cases, a linear relationship between absorbance (or fluorescence intensity) and the concentration of monosubstituted guanidine was observed. The results obtained by the procedures of Van Pilsum et al. [11] and Pesez and Bartos [12] and by the fluorimetric method [12] show reasonable correspondence (_+ 10%) to the actual concentrations of monosubstituted guanidines employed in the experiment. Such is not the case in the determinations involving Weber's [3] and Messineo's [13] modifications of the Sakaguchi procedure. The applicability of the various Sakaguchi methods to the determination of the arginine content of intact proteins was studied. Table 2 compares the arginine content of the protein solutions obtained with the various colorimetric and fluorimetric procedures with values obtained by automated amino acid analysis of acid hydrolysates of these same protein solutions. A linear relationship between the concentration of protein and absorbance was noted with the procedures of Weber
273
0.8
0.6 Z
o ,~ 0.4
'\
/¢ t
¢ /
\
0.2
,
i
480
,
i
500
,
i
,
520
i
540
,
560
A (nm)
Fig. 3. Absorbance spectra of monosubstituted guanidines obtained using Sakaguchi procedure I. The samples arginine (. . . . . . ) and guanidobutyric acid ( ) were present at a concentration of 0.3 ktmol in the assay.
TABLE 2 DETERMINATION OF THE ARGININE CONTENT OF INTACT PROTEINS BY VARIOUS MODIFICATIONS OF THE SAKAGUCHI PROCEDURE Procedures I - V are as described in Methods. Automated amino acid analysis performed on aliquots after acid hydrolysis (6 M HCI, 24 h, in vacuo) according to the procedure described in Ref. 10. The expected number of arginine residues per molecule is based on the reported amino acid compositions of the proteins as described in the references provided in the table. Protein
Residues per molecule I
II
III
IV
V
Amino acid Expected Reference analysis
~bonuclease 4.2±0.1 1 . 2 ± 0 . 1 0 . 9 ± 0 . 1 1.9±0.1 6.1±0.1 3.9 Lysozyme 10.3±0.5 7.3±0.1 6.2±0.2 9.4± 1.1 7.7±0.5 9.9 a-Chymot~psin 4.3±0.3 2.4±0.1 1.6±0.1 2.8±0.3 3.6±0.1 2.8 ChymotrypsinogenA 5.0±0.2 2.8±0.1 1.4±0.1 3.4±0.2 3.4±0.1 3.6 Trypsin 2.6±0.2 1.3±0.1 1.0±0.1 1.6±0.2 3.5±0.1 1.9 SubtiHsin 3.7±0.3 1.6±0.1 1.3±0.1 2.0±0.2 4.3±0.1 1.9 Thyrotropin 3.6±0.2 2.1±0.1 1.9±0.1 3.0±0.5 5.4±0.1 3.6
4.0 11.0 3.0 4.0 2.0 2.0 4.0
18 19 6 6 20 21 21
274 [3], Van Pilsum et al. [1 1], and Pesez and Bartos [12]. The method of Messineo [13] and the fluorimetric phenanthrenequinone procedure [12] failed to yield such a relationship.
Discussion
N2-Succinylarginine was selected to serve as a model to elucidate the unusual reactivity of an arginine residue located in the hexapeptide segment corresponding to the sequence 149-154 of chymotrypsin, towards the Sakaguchi reaction. This arginine gives approximately twice the color yield as compared to unmodified arginine in Weber's modification [3] of the Sakaguchi procedure [4]. N 2Acetylarginine, upon reaction with these same reagents, also gave nearly twice the amount of colored product as that obtained with an equivalent amount of arginine. Subsequent work indicated that a wide variety of monosubstituted guanidines exhibited this feature. Compounds similar in structure to arginine, but for acylation or elimination of the c~-amino group, gave increased absorptivity upon reaction with the Sakaguchi reagents as described by Weber [3]. The enhanced color given by N2-acylarginine derivatives appears to be due, in part, to stabilization of the colored complex formed upon reaction of the guanidine derivative with the Sakaguchi reagents. In addition, the colored product produced by the acylated compounds possesses different spectral characteristics than the corresponding arginine chromophore. Arginine derivatives showing enhanced color with Weber's reagents failed to exhibit significant enhancement when tested by several other modifications of the Sakaguchi procedure. This may indicate that the enhanced color noted with the Weber method is an inherent anomaly of that procedure. Thus, the standard curves prepared with arginine cannot be used to determine the concentrations of solutions of other monosubstituted guanidines when Weber's modification [3] of the Sakaguchi procedure is employed. Several reports have suggested that the Sakaguchi procedure may be applicable to the estimation of arginine in intact proteins [1,13], whereas others have stated that the method is unsuitable [16]. The current investigations tend to support this latter opinion. Surprisingly, Weber's modification [3] of the Sakaguchi procedure proved to be the most accurate, when tested with proteins such as ribonuclease and lysozyme. Serine proteases, however, indicate the presence of an additional Sakaguchi-positive component, in agreement with previous findings [1,5]. The demonstration, by this procedure, of an additional Sakaguchi positive component in the hexapeptide fragment 149-154 of chymotrypsin is consistent with similar observations recorded with N2-acyl arginine derivatives. The C-terminal location of the arginine in the hexapeptide does not appear to be responsible for the anomaly in view of the manifestation of the phenomenon in intact chymotrypsin. Furthermore, neither of the two arginine residues in trypsin, a serine protease which has also been shown to possess an additional Sakaguchi-positive component [1], is preceded by an aspartic acid residue. Hence, it is difficult to attribute the additional
275 S a k a g u c h i - p o s i t i v e c o m p o n e n t in c h y m o t r y p s i n to the A s p - A r g sequence p r e s e n t in the h e x a p e p t i d e segment. Thus, the a d d i t i o n a l Sakaguchi c o m p o n e n t a p p a r e n t with W e b e r ' s p r o c e d u r e a p p e a r s to be an inherent feature of serine proteases for as yet u n k n o w n reasons. The other m o d i f i c a t i o n s of the Sakaguchi p r o c e d u r e tested d i d n o t yield reliable estimates of the arginine c o n t e n t of a n y of the p r o t e i n s e m p l o y e d in these studies. The fluorimetric p h e n a n t h r e n e q u i n o n e p r o c e d u r e used [12] also d i d not give reliable values for the arginine content of proteins. S m i t h a n d M a c Q u a r r i e [17] have recently shown that, with suitable modifications, this p r o c e d u r e can yield reliable estimates of the arginine c o n t e n t of intact proteins. However, o p t i m a l reaction c o n d i t i o n s m u s t be d e t e r m i n e d for each p r o t e i n tested. This d i s a d v a n t a g e is offset s o m e w h a t b y the reliability of the m e t h o d when c o m p a r e d with those a t t a i n a b l e in p r o c e d u r e s discussed in the present report. In addition, the fluorimetric p r o c e d u r e is c o n s i d e r a b l y m o r e sensitive than the Sakaguchi method.
Simplified description of the method and its applications The applicability of the various modifications of the Sakaguchi procedure for the determination of arginine residues in intact proteins was investigated. The results obtained were compared with those determined by the ion-exchange chromatography of the HC1 hydrolysates of proteins. These studies suggest that none of the modifications of the Sakaguchi procedure is suitable for the determination of the arginine content of proteins.
Acknowledgement T h e research was s u p p o r t e d b y the N a t u r a l Sciences a n d Engineering R e s e a r c h C o u n c i l of C a n a d a .
References 1 Tomlinson, G. and Viswanatha, T. (1974) Anal. Biochem. 60, 15-24 2 Martin, B.M., Fliss, H., Rama Murthy, J., Stepanik, T. and Viswanatha, T. (1975) Life Sci. 17, 1265-1268 3 Weber, C.J. (1930) J. Biol. Chem. 86, 217 4 Sakaguchi, S. (1925) J. Biochem. (Tokyo) 5, 25-31 5 Rickert, W.S. and Viswanatha, T. (1967) Biochem. Biophys. Res. Commun. 28, 1028-1033 6 Dixon, G.H. and Neurath, H. (1957) J. Biol. Chem. 225, 1040-1059 7 Fliss, H. (1977) Ph.D. Thesis, University of Waterloo; Stepanik, T.M. (1977) Ph.D. Thesis, University of Waterloo, Ontario 8 Greenstein, J.P. and Winitz, M. (1961) Chemistry of the Amino Acids, Vol. 3, p. 1850. Wiley, New York 9 Yang, P.S. and Rising, M.M. (1931) J. Am. Chem. Soc. 53, 3183-3184 10 Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 11 Van Pilsum, J.F., Martin, R.P., Kito, E. and Hess, J. (1956) J. Biol. Chem. 222, 225-236 12 Pesez, M. and Bartos, J. (1974) Colorimetric and Fluorometric Analysis of Organic Compounds and Drugs. Marcel Dekker, New York
276 13 14 15 16 17 18 19 20
Messineo, L. (1966) Arch. Biochem. Biophys. 117, 534-540 Yamada, S. and Itano, H.A. (1966) Biochim. Biophys. Acta 130, 538-540 Itano, H.A., Hiroto, K., Kawaski, I. and Yamada, S. (1976) Anal. Biochem. 76, 134-141 Izumi, Y. (1965) Anal. Biochem. 12, 1-7 Smith, R.E. and MacQuarrie, R. (1978) Anal. Biochem. 90, 246-255. Sherwood, L.M. and Potts, J.T. Jr. (1965) J. Biol. Chem. 240, 3799-3805 Steiner, R.F. (1964) Biochim. Biophys. Acta 79, 51-63 Walsh, K.A., Kauffman, D.L., Sampath Kumar, K.S.V. and Neurath, H. (1964) Proc. Natl. Acad, Sc U.S.A. 51,301-308 21 Croft, L.R. (1973) Handbook of Protein Sequences. Joynson-Bruvers, Oxford