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Spectrophotometric determination of N-acetylmuramic acid in complex molecules
Analytica Chimica Acta, 242 (1991) 221-224 Elsevier Science Publishers B.V., Amsterdam
221
Spectrophotometric determination of N-acetylmuramic acid in complex molecules Ljubica
SesartiC
Department of Radioimmunology, Institute of Immunology, P.O. Box 266, 41000 Zagreb (Yugoslavia)
Olga Hadiija
*
Departmen: of Physical Chemistry, “Ruder BoSkoviC” Institute, 41000 Zagreb (Yugoslavia) (Received
17th April 1990)
Abstract A method for the determination molecules is presented. The hydrolysis Keywords: Acetylmuramic
of N-acetylmuramic acid in both synthetic of the sample and photometric determination
occurring complex in one step.
acid
Muramic acid {2-amino-3-O-[( R)-l-carboxyethyl]-2-deoxy-D-glucopyranose} is usually determined either as an amino sugar [l] or via the amino group with ninhydrin. These methods are also applicable to the determination of Nacetylmuramic acid after its deacetylation but, as they are not specific, prior separation from components giving similar reactions is needed. Specific determinations of muramic and N-acetylmuramic acid are based on the reaction of the lactic acid moiety of the molecule. These determinations can be performed after the release of lactic acid either by acid [2-51 or alkali [6,7] hydrolysis or by applying enzymatic treatment with D-lactate dehydrogenase [8]. Previously, a method for the determination of muramic acid, based on acid degradation of the molecule to acetaldehyde, which is then measured photometrically after coupling with p-hydroxydiphenyl, was elaborated [2]. The method was applied to the determination of free muramic acid in mixtures containing the components of bacterial cell wall peptidoglycan [9]. 0003-2670/91/$03.50
and naturally are performed
0 1991 - Elsevier Science
Publishers
This method has now been improved by modifying the amount of sulphuric acid, by raising the reaction temperature and by introducing hydrolysis of the sample and photometric determination of N-acetylmuramic acid in one step. The improved method is simpler and faster but its accuracy is retained.
EXPERIMENTAL
Materials Abbreviations of the compounds considered are given in Table 1. The following solutions were used: M (Sigma, St. Louis, MO), 1 mg ml-’ aqueous; PHD (BDH, Poole), recrystallized before use, m.p. 166-168OC, 1.5% (w/v) in 96% ethanol; concentrated sulphuric acid (Merck, Darmstadt); CuSO,. 5H,O (Kemika, Zagreb), 4% aqueous; GM (Institute of Immunology, Zagreb), 1 mg ml-’ aqueous; PGM (Pliva, Zagreb), 2 mg ml-’ aqueous; MDP (“Ruder BoSkoviC” Institute, Zagreb), 1 mg ml-’ aqueous; GMDP (“Ruder BoSkoviE” B.V.
1
used, their abbreviations
CHrORr
Structure
Compounds
TABLE
Name iV-Acetylmuramic acid Muramyl dipeptide
Disaccharide Disaccharide dipeptide Peptidoglycan monomer p-Hydroxydiphenyl
GM GMDP PGM PHD
their origin
M MDP BzlAcMAlaOMe BzlAcMAlaGlyOBzl BzlAcMAlaiGln0Br.l
concerning
Abbreviation
and references
R=OH R=L-Ala-o-iGln R=L-Ala-o-iGh-
meso-( r-NH,)-A,pm-D-Ala-o-Ala
R=OH, R,=R,=R,=H R=t_-Ala-o-iGln, R,=R,=R,=H R=L-Ala-OMe, R,=R,=Ac, R,=Bzl R=r_-Ala-Gly-OBzl, R,=H, R,=Ac, R,=Bzl R=t_-Ala-o-iGln-OBzl, R,=H, R,=Ac, R,=Bzl
Substituents
9, 14, 15 16 17-19
10,ll 12a 13 12b
Ref.
DETERMINATION
OF N-ACETYLMURAMIC
ACID
IN COMPLEX
MOLECULES
Institute), 1 mg ml-’ aqueous; BzlAcMAlaGlyOBzl (“Ruder BoSkoviE” Institute), 2.5 mg ml-’ in 50% ethanol; BzlAcMAlaOMe (“Ruder BoSkoviE” Institute), 2.5 mg ml-’ in 50% ethanol; BzlAcMAlaiGlnOBzl (“Ruder BoSkoviE” Institute), 5 mg ml-’ in 50% ethanol. Procedure A solution containing lo-40 pg of M was made up to 0.5 ml with 1 M sulphuric acid and hydrolysed at 100-105°C for 2 h. For conversion of lactic acid to acetaldehyde, after cooling, 5.5 ml of concentrated sulphuric acid were cautiously added, the tube was shaken vigorously, well stoppered with a ground glass stopper and heated for 30 min at 110-115°C. For colour development, after heating the tube was cooled under running water and 0.05 ml of copper(I1) sulphate solution and 0.1 ml of PHD solution were added. The tube was stoppered again, shaken and kept at 30” C for 30 min. The absorbance was measured at 560 nm in a PerkinElmer Lambda 3 UV-visible spectrophotometer against a reagent blank.
RESULTS
AND
DISCUSSION
The determination of N-acetylmuramic acid showed that the absorbance was proportional to concentration in the range lo-40 pg. The corresponding calibration graph obeyed the equation y = 0.0164x + 0.006 (r = 0.9999; n = 4). It was found earlier [2] that the reaction conditions proposed for the determination of muramic acid are
TABLE
2
Determination acetylmuramic Sample
of complex molecules containing acid under different experimental conditions Taken ( pg)
Hydrolysis 0 Reaction 120 Found
MDP PGM
50.0 100.0
29.4 79.1
(min) 30 (min)
120
30
30
( pg) 29.4 87.7
50.4 101.5
N-
TABLE
223 3
Determination
of complex
molecules
via N-acetylmuramic
Sample
Taken (pg)
Found
MDP BzlAcMAlaOMe BzlAcMAlaGlyOBzl BzlAcMAlaiGlnOBzl GM GMDP PGM
50.0 50.0 50.0 50.0 50.0 50.0 100.0
50.4 49.8 50.4 49.8 49.9 41.4 101.5
a Means
(pg) ’
acid
R.s.d. (W) 5.9 5.6 2.3 5.8 5.8 5.2 4.4
of four determinations.
satisfactory for releasing the lactic acid moiety and its conversion to acetaldehyde. These conditions are also adequate for the determination of N-acetylmuramic acid as the free molecule or as a disaccharide (GM), but are not suitable for the determination of larger molecules containing the N-acetylmuramic acid moiety. For example, using the previous procedure [2] the recovery of MDP was 18.6% and that of PGM 29.5%. Even if a prolonged reaction time was applied, the results were not significantly better. Table 2 compares the results obtained by introducing the hydrolysis step in the procedure and by modifying the reaction time. It can be seen that by prolonging the hydrolysis and by adopting a reaction time of 30 min satisfactory results were obtained. Alkaline hydrolysis with 1 M sodium hydroxide solution of 1 M sulphuric acid gives the same results. In Table 3, results for the determination of the N-acetylmuramic acid moiety in different types of molecules are given. The data demonstrate that the hydrolysis of the compound, release of the lactic acid moiety, its conversion to acetaldehyde and photometric determination can be achieved in one procedure and in a relatively short time, permitting the analysis of both synthetic and naturally occurring complex compounds via N-acetylmuramic acid.
This research was supported by the Department of Radioimmunology, Institute of Immunology, Zagreb, and by the Council for Scientific Research of SR Croatia, Zagreb.
224 REFERENCES 1 G. Blix and E.W. Jeanloz, in R.W. Jeanloz (Ed.), The Amino Sugars, Vol. lA, Academic, New York, 1969, p. 247. 2 0. Hadfija, Anal. Biochem., 60 (1974) 512. 3 R.E. Strange and L.H. Kent, Biochem. J., 71 (1959) 333. 4 L.H. Kent and R.E. Strange, in R.L. Whistler and M.L. Wolfrom (Eds.), Methods in Carbohydrate Chemistry, Vol. 1, Academic, New York, 1962. p. 250. 5 Y. Asabe, S. Kojima, M. Suzuki and S. Takitani, Anal. B&hem., 79 (1977) 73. 6 J.M. Ghuysen, E. Bricas, M. Leyh-Bouille, M. Lashe and G.D. Shockman, Biochemistry, 6 (1967) 2607. 7 H.R. Perkins, Biochem. J., 102 (1967) 29~. 8 D.J. Tipper, Biochemistry, 7 (1968) 1441. 9 Z. Valinger, B. Lade% and J. TomaSiC, B&him. Biophys. Acta, 701 (1982) 63. 10 C. Merser, P. Sinayi and A. Adam, Biochem. Biophys. Res. Commun., 66 (1975) 1316. 11 M. PongraEiC, PhD Thesis, University of Zagreb, 1988, p. 100.
Lj. SESARTld
AND
0. HADilJA
12 M. PongraEiC, PhD Thesis, University of Zagreb, 1988, (a) p. 106, (b) p. 122. 13 D. KegleviC, M. Pongracik and D. Kantoci, Croat. Chem. Acta, 58 (1985) 569. 14 B. LadeSiC, J. TomaSiC, S. Kveder and I. HrSak. B&hem. Biophys. Acta, 678 (1981) 12. 15 J. TomaSiC, Lj. SesartiC, S.A. Martin, Z. Valinger and B. LadeSiC, J. Chromatogr., 440 (1988) 405. 16 D. Kantoci, D. Keglevii: and A.E. Derome. Carbohydr. Res., 186 (1989) 77. 17 D. KegleviC, B. Lade&C, 0. Hadrija, J. TomaSiC, Z. Valinger, M. Pokorny and R. Naumsky, Eur. J. Biochem., 42 (1974) 389. 18 D. KegleviC, B. Lade&C, J. TomaSiC, Z. Valinger and R. Naumski, Biochim. Biophys. Acta, 585 (1979) 273. 19 S.A. Martin, M.L. Karnovsky, J.M. Krueger, J.R. Pappenheimer and K. Biemann, J. Biol. Chem., 259 (1984) 12652.
221
Spectrophotometric determination of N-acetylmuramic acid in complex molecules Ljubica
SesartiC
Department of Radioimmunology, Institute of Immunology, P.O. Box 266, 41000 Zagreb (Yugoslavia)
Olga Hadiija
*
Departmen: of Physical Chemistry, “Ruder BoSkoviC” Institute, 41000 Zagreb (Yugoslavia) (Received
17th April 1990)
Abstract A method for the determination molecules is presented. The hydrolysis Keywords: Acetylmuramic
of N-acetylmuramic acid in both synthetic of the sample and photometric determination
occurring complex in one step.
acid
Muramic acid {2-amino-3-O-[( R)-l-carboxyethyl]-2-deoxy-D-glucopyranose} is usually determined either as an amino sugar [l] or via the amino group with ninhydrin. These methods are also applicable to the determination of Nacetylmuramic acid after its deacetylation but, as they are not specific, prior separation from components giving similar reactions is needed. Specific determinations of muramic and N-acetylmuramic acid are based on the reaction of the lactic acid moiety of the molecule. These determinations can be performed after the release of lactic acid either by acid [2-51 or alkali [6,7] hydrolysis or by applying enzymatic treatment with D-lactate dehydrogenase [8]. Previously, a method for the determination of muramic acid, based on acid degradation of the molecule to acetaldehyde, which is then measured photometrically after coupling with p-hydroxydiphenyl, was elaborated [2]. The method was applied to the determination of free muramic acid in mixtures containing the components of bacterial cell wall peptidoglycan [9]. 0003-2670/91/$03.50
and naturally are performed
0 1991 - Elsevier Science
Publishers
This method has now been improved by modifying the amount of sulphuric acid, by raising the reaction temperature and by introducing hydrolysis of the sample and photometric determination of N-acetylmuramic acid in one step. The improved method is simpler and faster but its accuracy is retained.
EXPERIMENTAL
Materials Abbreviations of the compounds considered are given in Table 1. The following solutions were used: M (Sigma, St. Louis, MO), 1 mg ml-’ aqueous; PHD (BDH, Poole), recrystallized before use, m.p. 166-168OC, 1.5% (w/v) in 96% ethanol; concentrated sulphuric acid (Merck, Darmstadt); CuSO,. 5H,O (Kemika, Zagreb), 4% aqueous; GM (Institute of Immunology, Zagreb), 1 mg ml-’ aqueous; PGM (Pliva, Zagreb), 2 mg ml-’ aqueous; MDP (“Ruder BoSkoviC” Institute, Zagreb), 1 mg ml-’ aqueous; GMDP (“Ruder BoSkoviE” B.V.
1
used, their abbreviations
CHrORr
Structure
Compounds
TABLE
Name iV-Acetylmuramic acid Muramyl dipeptide
Disaccharide Disaccharide dipeptide Peptidoglycan monomer p-Hydroxydiphenyl
GM GMDP PGM PHD
their origin
M MDP BzlAcMAlaOMe BzlAcMAlaGlyOBzl BzlAcMAlaiGln0Br.l
concerning
Abbreviation
and references
R=OH R=L-Ala-o-iGln R=L-Ala-o-iGh-
meso-( r-NH,)-A,pm-D-Ala-o-Ala
R=OH, R,=R,=R,=H R=t_-Ala-o-iGln, R,=R,=R,=H R=L-Ala-OMe, R,=R,=Ac, R,=Bzl R=r_-Ala-Gly-OBzl, R,=H, R,=Ac, R,=Bzl R=t_-Ala-o-iGln-OBzl, R,=H, R,=Ac, R,=Bzl
Substituents
9, 14, 15 16 17-19
10,ll 12a 13 12b
Ref.
DETERMINATION
OF N-ACETYLMURAMIC
ACID
IN COMPLEX
MOLECULES
Institute), 1 mg ml-’ aqueous; BzlAcMAlaGlyOBzl (“Ruder BoSkoviE” Institute), 2.5 mg ml-’ in 50% ethanol; BzlAcMAlaOMe (“Ruder BoSkoviE” Institute), 2.5 mg ml-’ in 50% ethanol; BzlAcMAlaiGlnOBzl (“Ruder BoSkoviE” Institute), 5 mg ml-’ in 50% ethanol. Procedure A solution containing lo-40 pg of M was made up to 0.5 ml with 1 M sulphuric acid and hydrolysed at 100-105°C for 2 h. For conversion of lactic acid to acetaldehyde, after cooling, 5.5 ml of concentrated sulphuric acid were cautiously added, the tube was shaken vigorously, well stoppered with a ground glass stopper and heated for 30 min at 110-115°C. For colour development, after heating the tube was cooled under running water and 0.05 ml of copper(I1) sulphate solution and 0.1 ml of PHD solution were added. The tube was stoppered again, shaken and kept at 30” C for 30 min. The absorbance was measured at 560 nm in a PerkinElmer Lambda 3 UV-visible spectrophotometer against a reagent blank.
RESULTS
AND
DISCUSSION
The determination of N-acetylmuramic acid showed that the absorbance was proportional to concentration in the range lo-40 pg. The corresponding calibration graph obeyed the equation y = 0.0164x + 0.006 (r = 0.9999; n = 4). It was found earlier [2] that the reaction conditions proposed for the determination of muramic acid are
TABLE
2
Determination acetylmuramic Sample
of complex molecules containing acid under different experimental conditions Taken ( pg)
Hydrolysis 0 Reaction 120 Found
MDP PGM
50.0 100.0
29.4 79.1
(min) 30 (min)
120
30
30
( pg) 29.4 87.7
50.4 101.5
N-
TABLE
223 3
Determination
of complex
molecules
via N-acetylmuramic
Sample
Taken (pg)
Found
MDP BzlAcMAlaOMe BzlAcMAlaGlyOBzl BzlAcMAlaiGlnOBzl GM GMDP PGM
50.0 50.0 50.0 50.0 50.0 50.0 100.0
50.4 49.8 50.4 49.8 49.9 41.4 101.5
a Means
(pg) ’
acid
R.s.d. (W) 5.9 5.6 2.3 5.8 5.8 5.2 4.4
of four determinations.
satisfactory for releasing the lactic acid moiety and its conversion to acetaldehyde. These conditions are also adequate for the determination of N-acetylmuramic acid as the free molecule or as a disaccharide (GM), but are not suitable for the determination of larger molecules containing the N-acetylmuramic acid moiety. For example, using the previous procedure [2] the recovery of MDP was 18.6% and that of PGM 29.5%. Even if a prolonged reaction time was applied, the results were not significantly better. Table 2 compares the results obtained by introducing the hydrolysis step in the procedure and by modifying the reaction time. It can be seen that by prolonging the hydrolysis and by adopting a reaction time of 30 min satisfactory results were obtained. Alkaline hydrolysis with 1 M sodium hydroxide solution of 1 M sulphuric acid gives the same results. In Table 3, results for the determination of the N-acetylmuramic acid moiety in different types of molecules are given. The data demonstrate that the hydrolysis of the compound, release of the lactic acid moiety, its conversion to acetaldehyde and photometric determination can be achieved in one procedure and in a relatively short time, permitting the analysis of both synthetic and naturally occurring complex compounds via N-acetylmuramic acid.
This research was supported by the Department of Radioimmunology, Institute of Immunology, Zagreb, and by the Council for Scientific Research of SR Croatia, Zagreb.
224 REFERENCES 1 G. Blix and E.W. Jeanloz, in R.W. Jeanloz (Ed.), The Amino Sugars, Vol. lA, Academic, New York, 1969, p. 247. 2 0. Hadfija, Anal. Biochem., 60 (1974) 512. 3 R.E. Strange and L.H. Kent, Biochem. J., 71 (1959) 333. 4 L.H. Kent and R.E. Strange, in R.L. Whistler and M.L. Wolfrom (Eds.), Methods in Carbohydrate Chemistry, Vol. 1, Academic, New York, 1962. p. 250. 5 Y. Asabe, S. Kojima, M. Suzuki and S. Takitani, Anal. B&hem., 79 (1977) 73. 6 J.M. Ghuysen, E. Bricas, M. Leyh-Bouille, M. Lashe and G.D. Shockman, Biochemistry, 6 (1967) 2607. 7 H.R. Perkins, Biochem. J., 102 (1967) 29~. 8 D.J. Tipper, Biochemistry, 7 (1968) 1441. 9 Z. Valinger, B. Lade% and J. TomaSiC, B&him. Biophys. Acta, 701 (1982) 63. 10 C. Merser, P. Sinayi and A. Adam, Biochem. Biophys. Res. Commun., 66 (1975) 1316. 11 M. PongraEiC, PhD Thesis, University of Zagreb, 1988, p. 100.
Lj. SESARTld
AND
0. HADilJA
12 M. PongraEiC, PhD Thesis, University of Zagreb, 1988, (a) p. 106, (b) p. 122. 13 D. KegleviC, M. Pongracik and D. Kantoci, Croat. Chem. Acta, 58 (1985) 569. 14 B. LadeSiC, J. TomaSiC, S. Kveder and I. HrSak. B&hem. Biophys. Acta, 678 (1981) 12. 15 J. TomaSiC, Lj. SesartiC, S.A. Martin, Z. Valinger and B. LadeSiC, J. Chromatogr., 440 (1988) 405. 16 D. Kantoci, D. Keglevii: and A.E. Derome. Carbohydr. Res., 186 (1989) 77. 17 D. KegleviC, B. Lade&C, 0. Hadrija, J. TomaSiC, Z. Valinger, M. Pokorny and R. Naumsky, Eur. J. Biochem., 42 (1974) 389. 18 D. KegleviC, B. Lade&C, J. TomaSiC, Z. Valinger and R. Naumski, Biochim. Biophys. Acta, 585 (1979) 273. 19 S.A. Martin, M.L. Karnovsky, J.M. Krueger, J.R. Pappenheimer and K. Biemann, J. Biol. Chem., 259 (1984) 12652.