BIOCHEMICAL
13, 23-27
MEDICINE
Isolation
(1975)
of N-Methylated
Physiological
Fluids
Basic and
Protein
Amino
Acids
from
Hydrolysates
ROMAN T. MARKIW Biochemistry
Research Laboratory, Veterans Martinsburg, West Virginia Received
December
Administration 25401
Center,
30, 1974
Various methylated amino acids have been found free in human plasma and urine. The concentrations of these compounds were not significantly influenced by the diet. Accumulated evidence is consistent with the hypothesis that these compounds are of endogenous origin and that the possibility of their reutilization in the body is small (1). These amino acids are most likely derived from the hydrolysis of methylated protein in vivo, since methylation of protein occurs subsequent to the formation of peptide bonds (2,3). Biological significance of methylation of proteins is not fully elucidated. However, since methylated amino acids are found in such highly specialized proteins as actin, myosin, cytochrome c, and histones (3), the determination of these substances in human urine may be important for the study of various pathological states. METHODS
The 24-hr adult-male urine specimens were refrigerated immediately after voidings and desalted the day that the collection ended. Solvent systems used were: l), n-BuOH-HOAc-H,O (4: 1: 1, by volume); 2), i-PrOH-HC02H-Hz0 (4: 1: 1, by volume): and 3), pyridineacetone-3 M NH,OH (50: 30: 25, by volume). The ion-exchange resins obtained from Sigma were 200-400 mesh, except where indicated otherwise. Melting points were determined on the hot stage and are corrected. N-Guanidino-methylated-arginines. NG,N’“and N”,N’;-dimethylarginine were prepared from methylated pseudourea and the copper complex of ornithine as published elsewhere (l), except the isolation of product from the reaction mixture was simplified by omitting addition of HOAc and thioacetamide. The reaction mixture was applied to a column of 4 X 13 cm of Dowex-50 (H+), and ornithine was eluted with 0.1 M NH,OH. The gradual increase of NH,OH concentration to 1.5 M eluted a small peak followed by dimethylarginine. Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
24 NG-Methylarginine Reporter (4).
ROMAN
T. MARKIW
was synthesized
by the procedure
of Corbin
and
di-p-hydroxyazobenzene-p’-sutfonate. This compound was prepared by the general method of Kurtz (5) using a methylation procedure according to Mazzetti (6). To the lyophilized copper complex prepared from L-lysine * HCl(183 mg; 1.O mmole) (7) was added 1.0 gm of NaHCO,, 30 of absolute MeOH and 5 ml of CHBI, The mixture then was stirred and refluxed for 4 hr. After concentration, the excess of NaHCO, was decomposed with 9 ml of Dowex-50 (H+), then trimethyllysine was eluted from resin with five volumes of 1.5 M NH,OH. It was purified by filtration through 0.5 ml of Dowex-1 (Cl-) and crystallized as N’,N’,N’-trimethyllysine di-p-hydroxyazobenzene-p’-sulfonate from water. Yield 460 mg, 62%; mp 226-227” with decomposition (reported 225-226”, cf. Ref. (1)). The product was identical with the salt of trimethyllysine prepared from acetyllysine (8) and with material isolated from urine by the mixed mp and by chromatographic properties. Hydrolysis. Proteins of animal origin were hydrolyzed in 6 N HCI at 110” for 20 hr in vacua or under N, atmosphere. The hydrolysates were concentrated to dryness and the residual acid removed on Amberlite IR-45 (OH-). Alkaline hydrolysis was performed with 2 N KOH for 16 hr at 104” in capped polypropylene vessels. The hydrolysate then was neutralized with 4 M HClO,. isolation of basic fraction. Desalting of urine was accomplished by modification of a previously described procedure (9). One-half of the 24-hr urine specimen was applied on a 2.2 X 30-cm column of Dowex5OW X8 (H+) (20-50 mesh). The resin was washed with 500 ml of water and the retained material was eluted with 500 ml of 1.5 M NH,OH and concentrated to dryness. For isolation of urinary imidazoles the residue was additionally applied to a 2 x S-cm column of Dowex-50 (H+), neutral amino acids were washed out with 500 ml of 0.1 M pyridine, and then the basic fraction was eluted with 100 ml of 1.5 M NH,OH. The basic fractions from hydrolysates were isolated in a similar manner. Separation of basic amino acids. Desalted urine or the basic fraction was applied on a 1.1 x 70-cm column of Dowex-SOW X8 (NH4+), and eluted with 2,000 ml of a linear gradient from 0.0-0.2 M NH,OH in IO-ml fractions. After collection of about 150 fractions, the reservoir with 0.2 M N&OH was substituted with 1.5 M NH,OH and elution was continued until emergence of arginine (Table 1). Presence of amino acids was revealed by ninhydrin reaction on paper. Concentrated solutions were used in case of minor quantities. The purity Nc,N’,N’-Trimethyllysine
N-METHYLATED
BASIC AMINO ACIDS TABLE
SEPARATION
25
1
AMINO ACIDS PROM 50% OF DIURNAL NORMAL URINEONA COLUMNOFDOWEX-SOW(NH,+)
OF BASIC
HUMAN
Rf values in paper
Fractions 3-10
1l-22 23-32 34-31 41-45 47-50 69-73 75-83 102-113
115-120 133-142 164-168 173-180 173-180 178-184 188-l% 188-l%
Amount isolated hx)
Compounds Imidazole amino acids Traces Imidazole amino acids Glucosylgalactosylhydroxylysine Galactosylhydroxylysine 5-Hydroxylysine Omithine N
13, 23-27
MEDICINE
Isolation
(1975)
of N-Methylated
Physiological
Fluids
Basic and
Protein
Amino
Acids
from
Hydrolysates
ROMAN T. MARKIW Biochemistry
Research Laboratory, Veterans Martinsburg, West Virginia Received
December
Administration 25401
Center,
30, 1974
Various methylated amino acids have been found free in human plasma and urine. The concentrations of these compounds were not significantly influenced by the diet. Accumulated evidence is consistent with the hypothesis that these compounds are of endogenous origin and that the possibility of their reutilization in the body is small (1). These amino acids are most likely derived from the hydrolysis of methylated protein in vivo, since methylation of protein occurs subsequent to the formation of peptide bonds (2,3). Biological significance of methylation of proteins is not fully elucidated. However, since methylated amino acids are found in such highly specialized proteins as actin, myosin, cytochrome c, and histones (3), the determination of these substances in human urine may be important for the study of various pathological states. METHODS
The 24-hr adult-male urine specimens were refrigerated immediately after voidings and desalted the day that the collection ended. Solvent systems used were: l), n-BuOH-HOAc-H,O (4: 1: 1, by volume); 2), i-PrOH-HC02H-Hz0 (4: 1: 1, by volume): and 3), pyridineacetone-3 M NH,OH (50: 30: 25, by volume). The ion-exchange resins obtained from Sigma were 200-400 mesh, except where indicated otherwise. Melting points were determined on the hot stage and are corrected. N-Guanidino-methylated-arginines. NG,N’“and N”,N’;-dimethylarginine were prepared from methylated pseudourea and the copper complex of ornithine as published elsewhere (l), except the isolation of product from the reaction mixture was simplified by omitting addition of HOAc and thioacetamide. The reaction mixture was applied to a column of 4 X 13 cm of Dowex-50 (H+), and ornithine was eluted with 0.1 M NH,OH. The gradual increase of NH,OH concentration to 1.5 M eluted a small peak followed by dimethylarginine. Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
24 NG-Methylarginine Reporter (4).
ROMAN
T. MARKIW
was synthesized
by the procedure
of Corbin
and
di-p-hydroxyazobenzene-p’-sutfonate. This compound was prepared by the general method of Kurtz (5) using a methylation procedure according to Mazzetti (6). To the lyophilized copper complex prepared from L-lysine * HCl(183 mg; 1.O mmole) (7) was added 1.0 gm of NaHCO,, 30 of absolute MeOH and 5 ml of CHBI, The mixture then was stirred and refluxed for 4 hr. After concentration, the excess of NaHCO, was decomposed with 9 ml of Dowex-50 (H+), then trimethyllysine was eluted from resin with five volumes of 1.5 M NH,OH. It was purified by filtration through 0.5 ml of Dowex-1 (Cl-) and crystallized as N’,N’,N’-trimethyllysine di-p-hydroxyazobenzene-p’-sulfonate from water. Yield 460 mg, 62%; mp 226-227” with decomposition (reported 225-226”, cf. Ref. (1)). The product was identical with the salt of trimethyllysine prepared from acetyllysine (8) and with material isolated from urine by the mixed mp and by chromatographic properties. Hydrolysis. Proteins of animal origin were hydrolyzed in 6 N HCI at 110” for 20 hr in vacua or under N, atmosphere. The hydrolysates were concentrated to dryness and the residual acid removed on Amberlite IR-45 (OH-). Alkaline hydrolysis was performed with 2 N KOH for 16 hr at 104” in capped polypropylene vessels. The hydrolysate then was neutralized with 4 M HClO,. isolation of basic fraction. Desalting of urine was accomplished by modification of a previously described procedure (9). One-half of the 24-hr urine specimen was applied on a 2.2 X 30-cm column of Dowex5OW X8 (H+) (20-50 mesh). The resin was washed with 500 ml of water and the retained material was eluted with 500 ml of 1.5 M NH,OH and concentrated to dryness. For isolation of urinary imidazoles the residue was additionally applied to a 2 x S-cm column of Dowex-50 (H+), neutral amino acids were washed out with 500 ml of 0.1 M pyridine, and then the basic fraction was eluted with 100 ml of 1.5 M NH,OH. The basic fractions from hydrolysates were isolated in a similar manner. Separation of basic amino acids. Desalted urine or the basic fraction was applied on a 1.1 x 70-cm column of Dowex-SOW X8 (NH4+), and eluted with 2,000 ml of a linear gradient from 0.0-0.2 M NH,OH in IO-ml fractions. After collection of about 150 fractions, the reservoir with 0.2 M N&OH was substituted with 1.5 M NH,OH and elution was continued until emergence of arginine (Table 1). Presence of amino acids was revealed by ninhydrin reaction on paper. Concentrated solutions were used in case of minor quantities. The purity Nc,N’,N’-Trimethyllysine
N-METHYLATED
BASIC AMINO ACIDS TABLE
SEPARATION
25
1
AMINO ACIDS PROM 50% OF DIURNAL NORMAL URINEONA COLUMNOFDOWEX-SOW(NH,+)
OF BASIC
HUMAN
Rf values in paper
Fractions 3-10
1l-22 23-32 34-31 41-45 47-50 69-73 75-83 102-113
115-120 133-142 164-168 173-180 173-180 178-184 188-l% 188-l%
Amount isolated hx)
Compounds Imidazole amino acids Traces Imidazole amino acids Glucosylgalactosylhydroxylysine Galactosylhydroxylysine 5-Hydroxylysine Omithine N
