a rapid chromatographic method for the determination of ω- n ...

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A rapid chromatographic method for the determination of w-N-methylarginine in protein and muscle tissues1 CONSTANTINOS G. ZARKADAS Research Brcirrch, Cianad~ Agric~!ture, Ottalolea, Ont., Cczncacr'cr K6A 0C6 Received May 1. 1978 Revised July 5, 1978 Zarkadas, C. G. (1978) A rapid chromatographic method for the determination of o-N-me- thylarginines in protein and muscle tissues. Can. /. l3iochc.m. 56,952-927 PI rapid and sensitive chromatographic method is described for the complete separation and determination of MG-methyiarginine, A'",An;-dimethylas-ginine, I&TG,N'"-dimethylarginine, and related compounds in protein and muscle tissue hydrolysates. This method is designed to be used both with conventional amino acid analyzers using a single column (I0 x 0.9 cm) of Duraglm type DC-6A resin at 5I0C, one buffer system (0.21 M sodium citrate pH 5.40), and one buffer Wow rate (70ml/h) and with the accelerated and fully automatic Beckman Spinco model 121M amino acid analyzer using a 10 x 0.28cm microcoiumn packed with the Beckman type AA-10 resin and operated with the same buffer at 7.9rnl/Ba. This proceda~re was successfully applied to the determination of o-A'-methylarginines in various animal tissues, and the presence of four as yet unidentified ninhydrin-positive compounds is reported. Zarkadas, C. G. (1978) A rapid chromatographic method for the determination of o-N-me- thylarginines in protein and muscle tissues. Can. J. Biochc3rn. 56,952-957 Nous dkcrivons m e mkthode chromatographique rapide et sensible qua pimet la siparation complkte et la determination de la N"-ramCtlaylargini~tl~ la l%I'~,N"-dimkthylarginine, la N';,SfG- dimkthylarginine et les composCs apparerrtks dans les hydrolysats protkiques et les hydrolg- sats de Bissu mus~ulaire. Cette rnethode p u t ktre ~akilisee avec les analy$eurs conventionnels d9acides amines comportant une seule coionne (10 x 0.9crn) de risine Durrurn de type BC-6A B 51°C. ban systkme tampon (citrate de sodium 0.21 M, pH 5.40) et une viaesse d'icoulement du tampon (7Omllh) et avec l'analyseur d'acides aminis de Beckman Sginco m d i l e 121M, corn- plktement automatique et plus rapide. Ge modkle comporte une microcoloinne (10 x 0.28crn) remplie avec la risine Beckman de type AA-BO et il opere avec le meme tampon qkni \'ecoule B 7.9ml/h. Cette technique a permis de determiner avec succes les o-N-mithylarginines dnns divers tissus animaux et Je dCcelea la prisence de quatre composks rCagissant positivement B la nynhydrine et encore non identifiks. [Traduit par le journal] The methylation of specific arginines in proteins has attracted considerable attention since the discovery by Paik and Kim (1-2) that certain arginines in nuclear his- tones from cancerous, adult, and foetal cells were found to be methylated in variable amounts (3-6) and that their methylation may play an important role in the regulation sf gene transcription (7). Among the various mammalian tissues studied, they found that the brain had the highest ABBREVIATIONS: The abbreviations used (see (1975) Bfo- chem. J. 149, 1-16) are as follows: Arg(Me), w-NG-methyl- arginine; Arg(sMe2), m-NG,M"-dimethylarginine; Arg(uMe2), (0-NG,NfG-dimethyiarginine. The superscript G refers to the site of methylatiom in the guamidino groups as follows: H2NG-C-NHCH2CH2CH2CH(NH2)COOH, II HNfc with s denoting symmetrical and u denoting unsymmetrical methylation. Lys(4QH), 5-hydroxylysine; aHdys(50H), ~610-5- hydroxylysine; protein (arginine) methyltransferase (EC 2.1.1.23). S-ademsyl-L-methionine:protein AT-methyltrans- ferase. 'Contribution No. 355 from the Food Research Institute, Canada Agriculture, Ottawa. Ont . , Canada. levels of enzyme protein (arginine) methyltrans- ferase (2-51, which is involved in the posttranslational methylation of the arginine at position 107 in the amino acid sequence of the encephalitogenic basic Al protein of the central nervous system (8-10). It is now recognized that many other highly speci:ilized mammalian and bacterial proteins (1 1-12) are also methylated by this widely distributed cytoplasmic enzyme (5). These studies were facilitated by the development of several chromatographic and electrophoretic methods for the analysis of co-hr-methylarginines ( 12- 15) and it has now been established that three chemically and structur- ally distinct w-N-rnethylarglnines, Arg(Me), Arg(~aMe,), and Arg(sMe,), exist in certain proteins (5, 161. How- ever, the various methods used for their determination in both protein hydrolysates (5, 12- 18) and physiological fluids (19-20) have proven laborious, and the complete separation of Psrg(Me1 from arginine has not been possible. This paper describes a rapid and sensitive method for the complete separation of all a-M-methylarginines and related compounds using either the Beckman type AA-10 or the Durmrn type DC-6A resin and a single-column methodology. This simple chrorntitographic method, Can. J. Biochem. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 11/11/14 For personal use only.

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Page 1: A rapid chromatographic method for the determination of ω-               N               -methylarginines in protein and muscle tissues

A rapid chromatographic method for the determination of w-N-methylarginine in protein and muscle tissues1

CONSTANTINOS G. ZARKADAS Research Brcirrch, Cianad~ Agric~!ture, Ottalolea, O n t . , Cczncacr'cr K6A 0C6

Received May 1. 1978

Revised July 5, 1978

Zarkadas, C. G. (1978) A rapid chromatographic method for the determination of o-N-me- thylarginines in protein and muscle tissues. Can. /. l3iochc.m. 56,952-927

PI rapid and sensitive chromatographic method is described for the complete separation and determination of MG-methyiarginine, A'",An;-dimethylas-ginine, I&TG,N'"-dimethylarginine, and related compounds in protein and muscle tissue hydrolysates. This method is designed to be used both with conventional amino acid analyzers using a single column (I0 x 0.9 cm) of Duraglm type DC-6A resin at 5I0C, one buffer system (0.21 M sodium citrate pH 5.40), and one buffer Wow rate (70ml/h) and with the accelerated and fully automatic Beckman Spinco model 121M amino acid analyzer using a 10 x 0.28cm microcoiumn packed with the Beckman type AA-10 resin and operated with the same buffer at 7.9rnl/Ba. This proceda~re was successfully applied to the determination of o-A'-methylarginines in various animal tissues, and the presence of four as yet unidentified ninhydrin-positive compounds is reported.

Zarkadas, C. G. (1978) A rapid chromatographic method for the determination of o-N-me- thylarginines in protein and muscle tissues. Can. J . Biochc3rn. 56,952-957

Nous dkcrivons m e mkthode chromatographique rapide et sensible qua p i m e t la siparation complkte et la determination de la N"-ramCtlaylargini~tl~ la l%I'~,N"-dimkthylarginine, la N';,SfG- dimkthylarginine et les composCs apparerrtks dans les hydrolysats protkiques et les hydrolg- sats de Bissu mus~ulaire. Cette rnethode p u t ktre ~akilisee avec les analy$eurs conventionnels d9acides amines comportant une seule coionne (10 x 0.9crn) de risine Durrurn de type BC-6A B 51°C. ban systkme tampon (citrate de sodium 0.21 M , pH 5.40) et une viaesse d'icoulement du tampon (7Omllh) et avec l'analyseur d'acides aminis de Beckman Sginco m d i l e 121M, corn- plktement automatique et plus rapide. Ge modkle comporte une microcoloinne (10 x 0.28crn) remplie avec la risine Beckman de type AA-BO et il opere avec le meme tampon qkni \'ecoule B 7.9ml/h. Cette technique a permis de determiner avec succes les o-N-mithylarginines dnns divers tissus animaux et Je dCcelea la prisence de quatre composks rCagissant positivement B la nynhydrine et encore non identifiks.

[Traduit par le journal]

The methylation of specific arginines in proteins has attracted considerable attention since the discovery by Paik and Kim (1-2) that certain arginines in nuclear his- tones from cancerous, adult, and foetal cells were found to be methylated in variable amounts (3-6) and that their methylation may play an important role in the regulation sf gene transcription (7). Among the various mammalian tissues studied, they found that the brain had the highest

ABBREVIATIONS: The abbreviations used (see (1975) Bfo- chem. J . 149, 1-16) are as follows: Arg(Me), w-NG-methyl- arginine; Arg(sMe2), m-NG,M"-dimethylarginine; Arg(uMe2), (0-NG,NfG-dimethyiarginine. The superscript G refers to the site of methylatiom in the guamidino groups as follows:

H2NG-C-NHCH2CH2CH2CH(NH2)COOH, II

HNfc with s denoting symmetrical and u denoting unsymmetrical methylation. Lys(4QH), 5-hydroxylysine; aHdys(50H), ~610-5- hydroxylysine; protein (arginine) methyltransferase (EC 2.1.1.23). S-ademsyl-L-methionine:protein AT-methyltrans- ferase.

'Contribution No. 355 from the Food Research Institute, Canada Agriculture, Ottawa. Ont . , Canada.

levels of enzyme protein (arginine) methyltrans- ferase (2-51, which is involved in the posttranslational methylation of the arginine at position 107 in the amino acid sequence of the encephalitogenic basic A l protein of the central nervous system (8-10). It is now recognized that many other highly speci:ilized mammalian and bacterial proteins (1 1-12) are also methylated by this widely distributed cytoplasmic enzyme ( 5 ) .

These studies were facilitated by the development of several chromatographic and electrophoretic methods for the analysis of co-hr-methylarginines ( 12- 15) and it has now been established that three chemically and structur- ally distinct w-N-rnethylarglnines, Arg(Me), Arg(~aMe,), and Arg(sMe,), exist in certain proteins (5, 161. How- ever, the various methods used for their determination in both protein hydrolysates (5, 12- 18) and physiological fluids (19-20) have proven laborious, and the complete separation of Psrg(Me1 from arginine has not been possible.

This paper describes a rapid and sensitive method for the complete separation of all a-M-methylarginines and related compounds using either the Beckman type AA-10 or the Durmrn type DC-6A resin and a single-column methodology. This simple chrorntitographic method,

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ZARKADAS 953

based on our previous work (211, is designed to be used with both conventional and fully automatic amino acid analyzers and requires about 2 h for a complete analysis. Evidence for the presence of three unknown basic amino acid peaks in acid hydrolysates of brain, testis, and dia- phragm tissues and one as yet unidentified ninhydrin- positive compound in the liver tissue is also presented.

Materials and Methods Ikfatericrls

The Dura-arm type B C d A spherical resin of 11.0 2 I.B,um particle size diameter, which is equivalent to Beckman types AA-20 or W-2 resins. was obtained in the sodium form from the Durrum Chemical Corporation, Palo Alto, CA. The Beckman type AA-I0 or type W-3 resins used on the microcolumn system were obtained in the sodium form as 9.0 ~ts 0.5,urn particle size diameter from Beckrnan Instruments, Inc., Palo Alto, CA. The unusual standard methylated basic amino acids, NG,NG-di- methyl-L-arginine, NG,N'"-dimethyl-di mine, and NG-me- thyl-~-arginine-bidp-hydroxyaaobenzene-p'-sulfonate) mono- hydrate, were purchased from Calbiochem, La Jolla, CA.

Amino Acid Calibration Stundards All of the unusual amino acid standards were supplied as

chromatog~aphically hornogenecsus compounds and were used without further purification. The preparation of amino acid calibration standards employed for peak identification and standardization of the instruments was carried out as previously described 421). The p-hydroxyazobenzene-y'-sulfoniite salt of Argdhge) which does not interfere in the analysis was not re- moved prior to use in the calibration mixture.

Prepnmtiott of'Tissue Hydrolystzres. The tissues and organs used were taken from mature bovine

animals of the Animal Research Institute's herd immediately after exsanguination and were minced, dried to constant weight in vaclpo (95- IWC), pulverized in an electric driven end-runner mill, and then passed through a 152-pm mesh sieve. Tripficate samples were hydrolysed under vacuum with glass-distilled constant-boiling HCI (6.OM) at 110°C for periods of 24, 48, 72, and 96 h as described previously (21).

Procrdures.for Amino Acid Anulyses Amino acid analyses were carried out on either aconventional

o r a fully automated amino acid analyzer using single-column methodology a s follows.

Merhod A-Beckman model B20B, 10 x 0.9cm column of Dura-urn type DC-QA resin, eluted with 0.21 M sodium citrate buRer pH 5.40 at 51°C, a flow rate of 70ml/h, and 120psi (827kN/m2). After the emergence of the neutral and acidic amino acids, the regular ninhydrin reagent was pumped into the effluent stream at 35 rnllh.

Method B-Beckman model 121M, 10 x 0.28cm column of Beckman type AA-10 resin, eluted with the same sodium citrate buffer and at the same temperature but at a buffer flow rate of 7.9 mUh. The w-IV-methylarginines were determined from a concentrated tissue or organ hydrolysate sample (equivalent to 0.2 to 0.5 mg/20pl) in order to obtain reasonably sized peaks for these components, and the dimethyl sulfoxide - ninhydrin rea- gent of Moore (22) was pumped into the effluent stream at 3.84 ml/h following the emergence of the neutral and acidic amino acids.

In both systems, the instruments were equipped with a Mod- ule control (Autolab Spectra-Physics GmbH, 61 Barrnstadt, West Germmy) and a companion Autolab system AA (Beckman methodology bulletins AA-TB-001 to AA-TB-014) for comput- ing peak concentrations.

Results and Discussion

The separation of the w-N-methylarginines was steadied under a variety of chromatographic conditions in order to obtain optimum resolution in a minimum of time. It was found that shorter columns packed with spherical resins of small particle size and uniform particle size distribution, such as Durrum DC-GA and Beckman AA-10, not only permitted faster flow rates but also ex- hibited high resolving power because equilibrium was attained more rapidly. Chromatography conducted at various temperatures. pH, and sodium ion concentra- tions of the eluting buffer indicated that operation of the smaller columns with 0.28 M sodium citrate buffer pH 5.40 at 51°C gave the best separation. Thus, the results obtained on the resolution of these basic anmino acids as a function of column length are shown in Fig. 1. Although optimum separation of all methylated arginines and re- lated compounds was achieved on a 20 x 8.9 cm Durrum DC-6A column, the results demonstrate that the shorter column (18 x 0.9 cm) chosen for method A (see LWebhods section) gave good separation and required a maximum of 3 h for a complete analysis. This method is very simple to use both as a rapid analytical procedure and a s a direct preparative method for isolating and characterizing these compounds.

Figure 2A shows the elution pattern obtained when a synthetic mixture of w-N-methylarginines and related compounds was analyzed by method A. Although the relative positions assigned to the Arg(Me), Arg(sMe,), and Arg(mdMe,) are consistent with those reported by other authors (5, 13-18), method A gives a complete separation of all of these unique compounds especially between Arg(Me) and arginine. Table 1 lists the elution times and operational color yields obtained relative to a color yield of 1 .O for L-norleucine. With the precision of the chromatographic procedure (100 & 3.0%), the repro- ducibility of 21 such determinations was excellent. It should be pointed out, however, that the relative color yields reported in Table 1 apply only to the particular instrument and absorption cells (6.6mm) used in this study and must be redetermined for each instrument and batch of ninhydrin employed in order to maintain the high precision and accuracy of the method.

For purposes of comparison, the typical chromato- graphic separations obtained from acid hydrolysates of bovine testis, bmin, diaphragm, and liver tissues by method A are shown in Fig. 2. These results clearly demonstrate the complete separation of all three methy- lated arginines (Fig. 2A) and the presence of four major as yet unidentified ninhydrin-positive peaks (Figs. 2B to 2E). The chromatogram of Fig. 2B shows the separation of the diastereoisomers of Lys(SQW), Arg(sMe2), and Arg(uMe,) from bovine testis hydrolysates and the pres- ence of two major unknown peaks designated as I and 111. Unknown No. 1 was completely separated as a discrete peak following the ammonia peak while unknown No. III emerged from the column (146.6 min) a s a shoulder on the trailing edge of arginine. In addition to all three methy- lated argines listed in Table 2 , this same unknown No. 111 was observed in the chromatograph of bovine brain tis-

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CAN. J . BIOC'HEM. VOI,. 56, 1978

ELUTION TIME (min)

FIG. 1. Resolution of basic amino acids as a function of column length. The Beckman model 120B amino acid analy =a- using cdurnns of0.9-crndiameterof Burrurn type DC-6A resin wasoperated with0.21 Msodiun~ citrate buffer pH 5.40at 51"Cand a b~aRer flow rate of 74) ml/h.

sue (Fig. 2C). Although unknown III is reminiscent of one of the unknown peaks reported earlier by Paik and Kim (1-21, its identity remains to be established. As may be seen in the chromatographs of Figs. 2D and 2E, certain of the o-AT-methylarginim were also found in the acid hydrolysates of both diaphragm and liver tissues. Moreover, it is shown that unknown II exists mainly in acid hydrolysates of the bovine diaphragm (Fig. 2D), while unknown IV occurred only in liver tissue. The results obtained for the o-AT-arginine content of these tissues are reported in Table 2 . The relative concentra- tions 08' all these unidentified ninhydrin-positive peaks separated by this method were calculated by assuming a color constant value equal to that of arginine. However, further investigations are required to ascertain the origin,

TABLE I . Elution times and operational coIor yields obtained from the separation of the basic

amino acids by method A

Elution Operational Amino acids times, min color yields

-

5-HydroxyHysine 39.1 albo-5-Hydroxylysine 41.2 Lysine 56.3 0.79 (k0.04) Histidine 66.8 0.73 ( f 8.02) Ammonia 81.3 0.87(fO,OB)

qsMe2) 106.8 0.21 (+_0.01) Arg (uMe2) 113.0 0.20 (f8.01) Arg (Me) 131.0 0.51 (k0.01) Arginine 138.2 0.69 (+6.01)

Unknown No. I 95.4 I1 127.4

HBL 146.6 IV 171.7

NOTE: The operational color yield3 are calculated relative to the observed constant for norleucine (C - %5.7), taken as 1.0 at 570 nm. Values in brackets denote standlard deviations of 21 determinations ( P c 0.01).

TABLE 2. Amino acid composition of bovine brain, testis, diaphragm, and liver tissues determined by method A

Bovine tissue, rnoI/105 g dried tissue

-- - -- -

Amino acids Brain Testis Diaphragm Liver

Histidine 10.14 8.34 3.49 9.2% Arg 0 .56 0.89 - 0.61 Arg (uMe2) 0.68 0.47 0.31 0.49 Arg ( M a 0.56 0.53 - 0.49 Arginine 15.22 26.51 10.24 19.40

Unknown No. I - 0.28 0.05 - IT - - 3.02 -

I11 4.71 2.76 - - IV - - - 3.41

NOTE: The relative ccrnccntrations of the unknown peaks were calculated by assuming a color constant (6) value equai to that of arginine (CA,, = 17.8).

identify, and precise functional role of these different unknown compounds.

The o-N-methylarginines were also completely sepa- rated by method B , and as shown in Fig. 3A, each of the methylated components of the synthetic mixture emerged as well-separated discrete peaks. By selecting a resin of smaller particle size and a microcolumn of smal- ler diameter (0.28cm) and by adjusting the linear flow rate in proportion to the cross-sectional area, increased sensitivity and higher resolution were achieved. This procedure is fully automatic, requires 2 h for a complete analysis, and has the added advantage that nanogram amounts of these unique compounds can be determined with an accuracy of + 3.0%. For comparison, Fable 3 lists the retention times and calibration factors obtained with this system. It should be noted, however, that such values were redetermined every fifth analysis since slight variations in the flow rate and color yields are inherent in

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ZARKADAS

On a 0.9 x 90 cm coiurnn of Durrurn type BC-BA resin on Mdel 1208 (0.21~[~a+l citrate buffer, pH 5.400 3 0.01) at 51 'C

A. Synthetic Calibration Mixture

O. Diaphragm Tissue

E. L~ver Tissue

Unknown

I I 1 1 I I I

40 661 80 1861 1 20 148 160 180

TIME (rnin)

FIG. 2. Typical separation of basic amino acids and chromatographic analysis of bovine tissue hydrolysates using method 4. The upper two curves show absorbance at 590nm and the lower curve at 440nm.

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9.5 6 CAN. J . BIOCHEM. VOL. 56. I978

'ool 1" 1 Neutral A 0.28 x 10 em ~olulpsn of Bmkmam type AA-10 resin on Model 121M

90 I 1

His

i w . r a ~ v ~ ~ ~ w e J wtuar= r rs l~~ru , p r 1 ~ . s v v - W.WI , a t ra I

and Acidic Amino Acids

A. Synthetic Calibration Mixture

FIG. 3 . Chrornatograpbaic separation of basic amino acids and analysis of bovine diaphragm tissue using method B. The upper curve shows absorbance at 570 nm and the lower cuave at 440 nm.

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TABLE 3. Retention times and calibration factors amino acid analyzers; and to John P. Emery for technical (KF) obtained from the separation of basic amino assistance.

acids by method B

Retention Calibration Amino acids times, min factors (KF)

Tyrosine 12.6 Phenylalanine 13.0 5-Hydroxylysine 39.7 allo-5-HydroxyIysine 41 . 2 Lysine 50.7 Histidine 57.7 Ammonia 74.2 Arg ($Mez) 95.8 Arg (uMe2) 101 - 5 Arl3 (Me) 107.1 Arginine 122.4 Unknown PI I11 . 9

NOTF: Values for K F are the observed calibration factors per planomole ninhydrin-positive compound (KF in area (in ODs)/KF x - nmol). The symbol A denotes the area output in ODs.

the high-speed microanalyses. The application of this rapid and sensitive method was then tested with acid hydrolysates of diaphragm tissue (Fig. 3B) and large amounts of unknown I1 and the diastereoisomers of Lys(58H) were present in this tissue, but only trace amounts s f the o-N-methylarginines were detected. From the foregoing results, it is evident that this simple microcolumn chromatographic system can be conve- niently used as a rapid analytical method for the determi- nation of these unusual basic amino acids in protein or muscle tissue hydrolysates, especially when normal and pathological conditions are being compared.

The author Is indebted to Mrs. Mae Cox for typing this manuscript: to the Analytical Chemistry Services, Chemistry and Biology Research Institute, Canada De- partment of Agriculture, Ottawa, Ont., for the use of the

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