- Email: [email protected]
[4] Determination of sialic acid using an amino acid analyzer
48
ANALYTICAL METHODS
[4]
pyruvate and a-ketobutyrate are determined by adding known quantities of the a-keto acids to a duplicate sample before hydrolysis. Comments. As determined by this assay procedure, close to stoichiometric amounts of pyruvate and a-ketobutyrate are recovered, based on the loss of galactosamine and increase in absorbance at 240 nm. Typical results are presented in the table. Furthermore, the ratio of pyruvate to a-ketobutyrate produced (1.4:1) is the same as the ratio of the hydroxyamino acids, serine to threonine, destroyed. Pyruvate and a-ketobutyrate from the acid hydrolyzate were characterized as the 2,4-dinitrophenylhydrazone derivative,9 prepared by a modification of the method of Kun and Garcia-Hernandez. l° In addition, alanine and a-aminobutyrate are formed from the respective 2,4-dinitrophenylhydrazone derivatives by catalytic hydrogenolysis.11,~2 ' H. Katsuki, T. Yoshida, C. Tanegashima, and S. Tanaka, Anal. Biochem. g4, 112 (1968). 1oE. Kun and M. Garcia-Hernandez, Biochim. Biophys. Aeta 23, 181 (1957). 1~H. C. Brown and C. Brown, J. Amer. Chem. Soe. 84, 1495, 2829 (1962). F. Downs and W. Pigman, Biochemistry 8, 1760 (1969),
[4] D e t e r m i n a t i o n of Sialic A c i d U s i n g a n A m i n o Acid Analyzer 1 B y TEH-YUNG LIu
Sialic acids are acylated derivatives of an aminodeoxynonulosonic acid called neuraminic acid, 5-amino-3,5-dideoxy-D-glycero-D-galactononulosonic acid, The free amino compound does not occur in nature and has not yet been synthesized. In sialic acid isolated from biological material, the amino group is always substituted by acetyl or glycoyl radicals. Some natural sialic acids contain O-acetyl groups in addition. Sialic acids are present in erythrocytes, in various serum proteins, in glycoproteins, mucoproteins, and in various bacteria, 2 notably some strains of Escherichia colis and meningococeus.4 1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission and by a contract with the U.S. Army Medical Research and Development Command, Office of the Surgeon General (MIPR 9959). "~G. F. Springer (Ed.), Filth Macy Conference on Polysaccharides in Biology, Josiah Macy Jr. Foundation, New York, June I-3 (1959). ' G. T. Barry and W. F. Goebel, Nature (London) 179, 206 (1957).
[4]
DETERMINATION OF SIALIC ACID
49
Numerous colorimetric reactions for the sialic acid have been developed, including the Bial orcinol, 5 resorcinol, diphenylamine, 6 direct Ehrlich, 7 and tryptophan-perchloric acid reactions, and a method involving periodate oxidation and coupling with 2-thiobarbituric acid which has been used for the detection of 2-deoxyribose and 2-keto-3-deoxysugar acid. s-ix Most of the reactions are used for tile determination of other carbohydrates as well and cannot be applied directly to tissues or mixtures containing other carbohydrates. The Bial orcinol reaction gives identical colors with ketohexoses and sialic acid and can be used only when these substances are present in minimal quantity. More recently, Reinhold et al. ~- utilized the gas-liquid chromatographic procedure of Sweeley et al. ~3 for the determination of sialic acid in porcine ribonuclease as its trimethylsilylated methyl glycoside. The method to be described utilizes 2 N methanesulfonic acid as the catalyst for methanolysis to cleave glycosidic linkages and at the same time removes the amino acyl group from sialic acid. The amino acid analyzer is used for the quantitative estimation of sialic acid as methoxyneuraminic acid. The method in its present form has been applied to several polymers of sialic acids, glycoproteins, and bird's nest and was found to be successful in the determination of sialic acid in these samples. 4,~4
Methods Methanesulfonic acid ( 2 N ) in anhydrous methanol is prepared by pipetting 0.65 ml of methanesulfonic acid (reagent grade, Eastman Kodak) into a 5.0-ml volumetric flask, it is brought to volume with anhydrous methanol (reagent grade absolute methanol which has been soaked with Molecular Sieves, Fisher M-514, 10 g/100 ml of liquid). Sialic A c i d Standard. A standard solution containing 2.5 t~moles sialie acid (purchased from Pierce Chemical Co., Rockford, Illinois and furReagents.
4T. Y. Liu, E. C. Gotschlich, F. T. Dunne, and E. K. Jonsson, J. Biol. Chem. 246, 4703 (1971). SL. Svennerholm, Ark. Kemi 10, 577 (1957). 'A. Saifer and H. A. Siegel, J. Lab. Clin. Med. 53, 474 (1959). ~I. Wemer and L. Odin, Acta Soc. Med. Upsal. 57, 230 (1952). 8V. S. Waravdekar and L. D. Saslaw, Bioehim. Biophys. Acta 24, 439 (1957). DA. Weissbach and J. Hurwitz, J. Biol. Chem. 234, 705 (1959). 1oD. Aminoff, Virology 7, 355 (1959). 11L. Warren, J. Biol. Chem. 2,34, 1971 (1959), V. N. Reinhold, F. T. Dunne, J. C. Wriston, M. Schwarz, L. Sarda, and C. H. W. Him, J. Biol. Chem. 243, 6482 (1968). 1~C. C. Sweeley, R. Bentley, M. Makita, and W. W. Well, J. Amer. Chem. Soc. 85, 2497 (1963). 1, T. Y. Liu and Y. H. Chang, manuscript in preparation.
50
ANALYTICAL METHODS
~]
ther recrystallized by the method of McGuire and Binkley 15) is lyophilized. This sample is used to check the procedure and to establish the color factor for methoxyneuraminic acid on a Beckman-Spinco Model 120C amino acid analyzer. Crystalline sample of methoxyneuraminic acid prepared according to the procedure of Klenk and Faillard 16 can also be used for this purpose. The color values obtained for both samples should agree within _+3%. Methanolysis and Analysis. Samples containing 0.1-2.5 ~moles of sialic acids are placed in glass tubes (Kimble 45066A, 12 × 150 mm) equipped with Teflon-lined screw caps. Methanesulfonic acid (2 N) in anhydrous methanol, 0.5 ml, is added, and the tubes are flashed with nitrogen and sealed with the cap. The tubes are immersed into a heating block (Exacta-Heat, Model 218, Techni Laboratory Instruments, Pequannock, New Jersey; hole depth 50 ram) maintained at 6 5 _ 1 ° for various lengths of time. At the end of each incubation period, the tubes are attached with a short section of Tygon to the condensor of a rotary evaporator which can be operated with the condensor axis at a downward tilt of about 30 °. The methanol is removed in about 20 minutes at 40 ° . Alternatively, the solvent can be removed by evaporation in a stream of nitrogen at 40 °. The latter procedure is more time consuming. The product of methanolysis, methyl (methyl D-neuraminid)ate [compound (II), Fig. 1], is converted to methoxyneuraminic acid [compound (III) Fig. 1] by saponification in the following manner. The methanolysate is treated with 1.10 ml of a 1.0N NaOH for 60 minutes at 25 ° (pit should be 12-13). The solution is transferred quantitatively to a 2.0-ml or a 5.0-ml volumetric flask and made up to volume with water. An aliquot (0.5-2.0 ml) of the sample is used for analysis on the 60-cm column of the amino acid analyzer with the pH 3.25 buffer as eluent. The amino acid analyzer constant for methoxyneuraminic acid determined with an authentic crystalline sample was 5.12 for an instrument for which the aspartic acid constant is 8.84. The elution volume of methoxyneuraminic acid and aspartic acid are 40 and 65 ml, respectively, on this instrument. Calculations. It is important to note that the rate of cleavage of glycosidic bonds involving sialic acids differs from one sample to the other and that some destruction of sialic acid is unavoidable during methanolysis; the degree of destruction being dependent upon the composition and the concentration of the sample used and the time of meth~5E. McGuire and S. B. Binkley, Biochemistry 3, 247 (1964). ~'E. Klenk and tI. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 298, 230 (1954).
[4]
DETERMINATION OF SIALIC ACID
H~
o
.~
HOI'~C H~
/CHs
. HO~, H
COOH
H // . . . . O" // C H s ~ N ~
51
o
HOH2C H""
methanolysis [H+]
COOCHs
+
// . . . . O" // H s N ~
(i)
(n)
INaOH
HO~. J-I
COOH
HOH2C ~¢C~/ ~
/CHs
~.~
H'/"OH~o ~
"O--CH.
0/CHs
HO~. ~
,
HOHuC H":?'-OH~o///~COONa
(IV)
(III) (I) fl-Methoxy-N-acetylneuraminic acid
(II) fl-Methyl(methyI v-neuraminid)ate
(HI) fl-Methoxyneuraminic acid (IV) a-Methoxy-N-acetylneuraminie acid FIG. 1. structure of N-acetylneuraminic acid and its derivatives.
anolysis. A more accurate evaluation of the content of sialic acid is obtained from the study of multiple analyses with samples heated for varying periods of time. The values obtained at the two times of methanolysis, for instance, at 16 and 32 hours, are extrapolated to zero time assuming first-order kinetics.
Comments Successful analyses of amino sugars in glycoproteins or polysaccharides depends upon: (a) the complete cleavage of every glycosidic bond; (b) prevention of destruction of amino sugars during hydrolysis; and (c) quantitative procedure suitable for the characterization and determination of the amino sugar released. Such a condition seems to have been achieved by the combined use of methanolysis in 2 N methanesulfonic acid at 65 ° for the cleavage of glycosidic bonds and the amino acid analyzer column for the quantitative estimation of the released sialic acid. This conclusion is justified by the results of analyses of a number of sialic acid-containing glycoproteins and polysaccharides as shown in the table. Sialic acids can be liberated from glycoproteins or polysaccharides by three different procedures. These include: (1) mild acid hydrolysis
52
ANALrrICAL ~v.THODS
[4]
ANALYSES OF SIALIC ACIDS IN GLYCOPROTEINSAND POLYSACCHARIDESa Method of analyses
Samples
Methanolic methauesulfonic Neuramln~dases: 0.1 N H2S04: acid: Amino Warren test~ Warren test ¢ acid analyzer~
1. al-Glycoprotein (human) ~ 2. Submax mucin (sheep)," 3. Desialized submax mucin (sheepY 4. Bird's nest (swallow) 5. Escherichia coli colominic acidg 6. Meningococcal B-polysaccharideh 7. Meningococcal C-polysaccharideh 8. Group B streptococcus~
37.7 103.0 < 1.0
37.0 92.1 <:1.0
39.9 98.1 <1.0
31.2 245.0 263.0 < 2.5 < 1.0
30.1 75.0 84.0 125.0 52.9
40.1 273.1 260.0 269.0 98.7
Results are expressed as micromoles of sialie acid per 100 mg of samples. b Samples were digested with neuraminidase (Clostridium perfringens, obtained from Pierce Chemical Co.) at 37 ° for 24 hours essentially according to the method of Cassidy et al. [J. T. Cassidy, G. W. Jourdian, and S. Roseman, J. Biol. Chem. 240, 3501 (1965)]. c The conditions used were 0.1 N H2S04, 80 °, 1 hour (R. G. Spiro, Vol. 8, p. 14). J See text for detail. The values reported were obtained by extrapolation of 16- and 32-hour values to zero time or infinite time, assuming first-order kinetics. • Obtained as a gift from Dr. E. A. Popenoe of Brookhaven National Laboratory. t Sheep submax mucin R-2 samples and its desialized product were obtained from Dr. S. Roseman of the Johns Hopkins University in 1962. a E. coli colominic acid was prepared from strain K235 by the method of Gotschlich. [E. C. Gotschlich, T. Y. Liu, and M. S. Artenstein, J. Exp. Med. 129, 1349 (1969)]. h T. Y. Liu, E. C. Gotschlich, F. T. Dunne, and E. K. Jonsson, J. Biol. Chem. 246, 4703 (1971). Obtained from Drs. Rebecca Lancefield and Emil C. Gotschlich of The Rockefeller University. w i t h 0.1 N H2S04 a t 80 ° for 1 hourlY; (2) t h e a c t i o n of n e u r a m i n i d a s e s ; a n d (3) a c i d - c a t a l y z e d m e t h a n o l y s i s w i t h a n h y d r o u s a c i d such as 2 N m e t h a n e s u l f o n i c acid. S i a l i c a c i d is in g e n e r a l u n s t a b l e in a q u e o u s acidic conditions. T h e s t a n d a r d m i l d c o n d i t i o n s (0.1 N H2SO4, 80 °, 1 hour) c o m m o n l y used a r e good p e r h a p s o n l y for t h e l i b e r a t i o n of sialic a c i d f r o m g l y c o p r o t e i n s b e c a u s e of its t e r m i n a l p o s i t i o n in these molecules. H o w e v e r , s t r o n g e r c o n d i t i o n s in a q u e o u s m e d i a will r a p i d l y cause c o m p l e t e d e s t r u c t i o n of s i a l i c acid. W h e n t h i s m e t h o d w a s a p p l i e d to p o l y m e r of sialic a c i d 1~R. G. Spiro, Vol. 8 [1].
[4]
DETERMINATION OF SIALIC ACID
53
with molecular weight in excess of 100,000 such as the meningococcal B- and C-polysaccharides and the E. coli colominic acid (see the table), the method failed to yield more than 20% of the sialic acid content in 60 minutes. Prolonged incubation (3-4 hours) resulted in higher recovery of sialic acid (30-50%), but the yield never exceeded 55% from both B- and C-polysaccharides of meningococcus.4 The failure of this procedure to yield more than 55% of the sialic acid from these polysaccharides is most likely caused by compensating factors; continuous release and destruction of the released sialic acid during the hydrolysis in 0.1 N H2S04 at 80 °. All or a large part of the sialic acid pre~ent in glycoproteins and polysaccharides can also usually be released by the action of neuraminidase (see the table). However, in some instances enzymatic hydrolysis of the sialic acid containing polysaccharide did not result in the release of any appreciable amount of sialic acid as are shown in the case of the C-polysaccharide from meningococcus4 and the polysaccharide isolated from a strain of group B streptococcus. Evidently, these neuraminidases are not capable of hydrolyzing certain glycosidic linkages of sialic acid or its O-acetylated derivatives. When 2 N methanesulfonic acid is used as a catalyst in methanolysis, the release of sialic acid from the polysaccharides or the glycoproteins has been consistently higher, as is shown in the table. For the estimation of sialic acid content in polysaccharide and glycoproteins, the advantages of using methanesulfonic acid as a catalyst for methanolysis, and the amino acid analyzer for the quantitative estimation of this amino sugar, are 3-fold. First, the reagent is effective in causing more complete cleavage of sialic acid from the polysaccharides and the glycoproteins. Second, the product of methanolysis after removal of solvent and saponification can be analyzed without further derivatization such as is required for the gas chromatographic procedure. Third, the product, methoxyneuraminic acid, is the most stable derivative of sialic acid and it is eluted at a unique position on the chromatogram of the amino acid analyzer column, which serves to identify this amino sugar. For the analysis of amino sugars in glycoproteins or polysaccharides, examination of the data as a function of hydrolysis time is important. It provides an opportunity to ascertain whether the particular amino sugar in question is stable under the condition of hydrolysis and if it has been completely liberated from the glycoproteins or the polysaccharides. It permits an extrapolation of the values, either to zero time or to infinite time to correct for the hydrolysis time on the destruction or release of amino sugars. This method of calculation prob-
54
ANALYTICXL METHODS
[S]
ably comes as close to giving accurate results as is possible at present. For this purpose at least two companion hydrolyzates, heated for 16 and 32 hours, are required. The values obtained at the two times of hydrolysis are extrapolated to give best figures.
[ 5 ] M o l e c u l a r W e i g h t D e t e r m i n a t i o n of G l y c o p r o t e i n s b y P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s in S o d i u m Dodecyl Sulfate B y JERE P. SEGREST a n d RICHARD L. JACKSON
Electrophoresis on polyacrylamide gels in the detergent sodium dodecyl sulfate [CH3(CH2)loCH2OSO3Na], abbreviated SDS, is a rapid and often employed technique for the determination of the molecular weights of proteins2 -3 The usefulness of this procedure for accurate molecular weight determinations depends upon two factors. (1) Proteins in general bind constant amounts of SDS per gram when saturated. 4,~ The protein then has an overall negative charge that masks its intrinsic charge, 2,3 resulting in a constant charge to mass ratio for proteins2 ,5 (2) Proteins saturated with SDS take on a rodlike configuration, the length of the structure being proportional to its polypeptide chain length, and thus its molecular weight? Principle This procedure is not directly applicable to molecular weight determinations of glycoproteins. Glycoproteins containing more than 1055 carbohydrate behave anomalously during SDS polyacrylamide gel electrophoresis when compared to standard proteins? ,r The cause of this anomalous behavior is a decreased binding of SDS per gram of glycoprotein as compared with standard proteins. 7 The lower SDS binding results in a decreased charge to mass ratio for glycoproteins versus I A. L. Shapiro, E. Vifiuela, and J. V. Maizel, Biochem. Biophys. Res. Commun. 28, 815 (1967). 2K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). s A. K. Danker and R. R. Rueekert, J. Biol. Chem. 244, 5074 (1969). "J. A. Reynolds and C. Tanford, Proc. Nat. Aead. Sei. U.8. 66, 1002 (1970). 5j. A. Reynolds and C. Tanford, J. Biol. Chem. 9,45, 5101 (1970). 8M. S. Bretscher, Nature (London) New Biol. 231, 229 (1971). J. P. Segrest, R. L. Jackson, E. P. Andrews, and V. T. Marehesi, Bioehem. Biophys. Res. Commun. 44, 390 (1971).
ANALYTICAL METHODS
[4]
pyruvate and a-ketobutyrate are determined by adding known quantities of the a-keto acids to a duplicate sample before hydrolysis. Comments. As determined by this assay procedure, close to stoichiometric amounts of pyruvate and a-ketobutyrate are recovered, based on the loss of galactosamine and increase in absorbance at 240 nm. Typical results are presented in the table. Furthermore, the ratio of pyruvate to a-ketobutyrate produced (1.4:1) is the same as the ratio of the hydroxyamino acids, serine to threonine, destroyed. Pyruvate and a-ketobutyrate from the acid hydrolyzate were characterized as the 2,4-dinitrophenylhydrazone derivative,9 prepared by a modification of the method of Kun and Garcia-Hernandez. l° In addition, alanine and a-aminobutyrate are formed from the respective 2,4-dinitrophenylhydrazone derivatives by catalytic hydrogenolysis.11,~2 ' H. Katsuki, T. Yoshida, C. Tanegashima, and S. Tanaka, Anal. Biochem. g4, 112 (1968). 1oE. Kun and M. Garcia-Hernandez, Biochim. Biophys. Aeta 23, 181 (1957). 1~H. C. Brown and C. Brown, J. Amer. Chem. Soe. 84, 1495, 2829 (1962). F. Downs and W. Pigman, Biochemistry 8, 1760 (1969),
[4] D e t e r m i n a t i o n of Sialic A c i d U s i n g a n A m i n o Acid Analyzer 1 B y TEH-YUNG LIu
Sialic acids are acylated derivatives of an aminodeoxynonulosonic acid called neuraminic acid, 5-amino-3,5-dideoxy-D-glycero-D-galactononulosonic acid, The free amino compound does not occur in nature and has not yet been synthesized. In sialic acid isolated from biological material, the amino group is always substituted by acetyl or glycoyl radicals. Some natural sialic acids contain O-acetyl groups in addition. Sialic acids are present in erythrocytes, in various serum proteins, in glycoproteins, mucoproteins, and in various bacteria, 2 notably some strains of Escherichia colis and meningococeus.4 1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission and by a contract with the U.S. Army Medical Research and Development Command, Office of the Surgeon General (MIPR 9959). "~G. F. Springer (Ed.), Filth Macy Conference on Polysaccharides in Biology, Josiah Macy Jr. Foundation, New York, June I-3 (1959). ' G. T. Barry and W. F. Goebel, Nature (London) 179, 206 (1957).
[4]
DETERMINATION OF SIALIC ACID
49
Numerous colorimetric reactions for the sialic acid have been developed, including the Bial orcinol, 5 resorcinol, diphenylamine, 6 direct Ehrlich, 7 and tryptophan-perchloric acid reactions, and a method involving periodate oxidation and coupling with 2-thiobarbituric acid which has been used for the detection of 2-deoxyribose and 2-keto-3-deoxysugar acid. s-ix Most of the reactions are used for tile determination of other carbohydrates as well and cannot be applied directly to tissues or mixtures containing other carbohydrates. The Bial orcinol reaction gives identical colors with ketohexoses and sialic acid and can be used only when these substances are present in minimal quantity. More recently, Reinhold et al. ~- utilized the gas-liquid chromatographic procedure of Sweeley et al. ~3 for the determination of sialic acid in porcine ribonuclease as its trimethylsilylated methyl glycoside. The method to be described utilizes 2 N methanesulfonic acid as the catalyst for methanolysis to cleave glycosidic linkages and at the same time removes the amino acyl group from sialic acid. The amino acid analyzer is used for the quantitative estimation of sialic acid as methoxyneuraminic acid. The method in its present form has been applied to several polymers of sialic acids, glycoproteins, and bird's nest and was found to be successful in the determination of sialic acid in these samples. 4,~4
Methods Methanesulfonic acid ( 2 N ) in anhydrous methanol is prepared by pipetting 0.65 ml of methanesulfonic acid (reagent grade, Eastman Kodak) into a 5.0-ml volumetric flask, it is brought to volume with anhydrous methanol (reagent grade absolute methanol which has been soaked with Molecular Sieves, Fisher M-514, 10 g/100 ml of liquid). Sialic A c i d Standard. A standard solution containing 2.5 t~moles sialie acid (purchased from Pierce Chemical Co., Rockford, Illinois and furReagents.
4T. Y. Liu, E. C. Gotschlich, F. T. Dunne, and E. K. Jonsson, J. Biol. Chem. 246, 4703 (1971). SL. Svennerholm, Ark. Kemi 10, 577 (1957). 'A. Saifer and H. A. Siegel, J. Lab. Clin. Med. 53, 474 (1959). ~I. Wemer and L. Odin, Acta Soc. Med. Upsal. 57, 230 (1952). 8V. S. Waravdekar and L. D. Saslaw, Bioehim. Biophys. Acta 24, 439 (1957). DA. Weissbach and J. Hurwitz, J. Biol. Chem. 234, 705 (1959). 1oD. Aminoff, Virology 7, 355 (1959). 11L. Warren, J. Biol. Chem. 2,34, 1971 (1959), V. N. Reinhold, F. T. Dunne, J. C. Wriston, M. Schwarz, L. Sarda, and C. H. W. Him, J. Biol. Chem. 243, 6482 (1968). 1~C. C. Sweeley, R. Bentley, M. Makita, and W. W. Well, J. Amer. Chem. Soc. 85, 2497 (1963). 1, T. Y. Liu and Y. H. Chang, manuscript in preparation.
50
ANALYTICAL METHODS
~]
ther recrystallized by the method of McGuire and Binkley 15) is lyophilized. This sample is used to check the procedure and to establish the color factor for methoxyneuraminic acid on a Beckman-Spinco Model 120C amino acid analyzer. Crystalline sample of methoxyneuraminic acid prepared according to the procedure of Klenk and Faillard 16 can also be used for this purpose. The color values obtained for both samples should agree within _+3%. Methanolysis and Analysis. Samples containing 0.1-2.5 ~moles of sialic acids are placed in glass tubes (Kimble 45066A, 12 × 150 mm) equipped with Teflon-lined screw caps. Methanesulfonic acid (2 N) in anhydrous methanol, 0.5 ml, is added, and the tubes are flashed with nitrogen and sealed with the cap. The tubes are immersed into a heating block (Exacta-Heat, Model 218, Techni Laboratory Instruments, Pequannock, New Jersey; hole depth 50 ram) maintained at 6 5 _ 1 ° for various lengths of time. At the end of each incubation period, the tubes are attached with a short section of Tygon to the condensor of a rotary evaporator which can be operated with the condensor axis at a downward tilt of about 30 °. The methanol is removed in about 20 minutes at 40 ° . Alternatively, the solvent can be removed by evaporation in a stream of nitrogen at 40 °. The latter procedure is more time consuming. The product of methanolysis, methyl (methyl D-neuraminid)ate [compound (II), Fig. 1], is converted to methoxyneuraminic acid [compound (III) Fig. 1] by saponification in the following manner. The methanolysate is treated with 1.10 ml of a 1.0N NaOH for 60 minutes at 25 ° (pit should be 12-13). The solution is transferred quantitatively to a 2.0-ml or a 5.0-ml volumetric flask and made up to volume with water. An aliquot (0.5-2.0 ml) of the sample is used for analysis on the 60-cm column of the amino acid analyzer with the pH 3.25 buffer as eluent. The amino acid analyzer constant for methoxyneuraminic acid determined with an authentic crystalline sample was 5.12 for an instrument for which the aspartic acid constant is 8.84. The elution volume of methoxyneuraminic acid and aspartic acid are 40 and 65 ml, respectively, on this instrument. Calculations. It is important to note that the rate of cleavage of glycosidic bonds involving sialic acids differs from one sample to the other and that some destruction of sialic acid is unavoidable during methanolysis; the degree of destruction being dependent upon the composition and the concentration of the sample used and the time of meth~5E. McGuire and S. B. Binkley, Biochemistry 3, 247 (1964). ~'E. Klenk and tI. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 298, 230 (1954).
[4]
DETERMINATION OF SIALIC ACID
H~
o
.~
HOI'~C H~
/CHs
. HO~, H
COOH
H // . . . . O" // C H s ~ N ~
51
o
HOH2C H""
methanolysis [H+]
COOCHs
+
// . . . . O" // H s N ~
(i)
(n)
INaOH
HO~. J-I
COOH
HOH2C ~¢C~/ ~
/CHs
~.~
H'/"OH~o ~
"O--CH.
0/CHs
HO~. ~
,
HOHuC H":?'-OH~o///~COONa
(IV)
(III) (I) fl-Methoxy-N-acetylneuraminic acid
(II) fl-Methyl(methyI v-neuraminid)ate
(HI) fl-Methoxyneuraminic acid (IV) a-Methoxy-N-acetylneuraminie acid FIG. 1. structure of N-acetylneuraminic acid and its derivatives.
anolysis. A more accurate evaluation of the content of sialic acid is obtained from the study of multiple analyses with samples heated for varying periods of time. The values obtained at the two times of methanolysis, for instance, at 16 and 32 hours, are extrapolated to zero time assuming first-order kinetics.
Comments Successful analyses of amino sugars in glycoproteins or polysaccharides depends upon: (a) the complete cleavage of every glycosidic bond; (b) prevention of destruction of amino sugars during hydrolysis; and (c) quantitative procedure suitable for the characterization and determination of the amino sugar released. Such a condition seems to have been achieved by the combined use of methanolysis in 2 N methanesulfonic acid at 65 ° for the cleavage of glycosidic bonds and the amino acid analyzer column for the quantitative estimation of the released sialic acid. This conclusion is justified by the results of analyses of a number of sialic acid-containing glycoproteins and polysaccharides as shown in the table. Sialic acids can be liberated from glycoproteins or polysaccharides by three different procedures. These include: (1) mild acid hydrolysis
52
ANALrrICAL ~v.THODS
[4]
ANALYSES OF SIALIC ACIDS IN GLYCOPROTEINSAND POLYSACCHARIDESa Method of analyses
Samples
Methanolic methauesulfonic Neuramln~dases: 0.1 N H2S04: acid: Amino Warren test~ Warren test ¢ acid analyzer~
1. al-Glycoprotein (human) ~ 2. Submax mucin (sheep)," 3. Desialized submax mucin (sheepY 4. Bird's nest (swallow) 5. Escherichia coli colominic acidg 6. Meningococcal B-polysaccharideh 7. Meningococcal C-polysaccharideh 8. Group B streptococcus~
37.7 103.0 < 1.0
37.0 92.1 <:1.0
39.9 98.1 <1.0
31.2 245.0 263.0 < 2.5 < 1.0
30.1 75.0 84.0 125.0 52.9
40.1 273.1 260.0 269.0 98.7
Results are expressed as micromoles of sialie acid per 100 mg of samples. b Samples were digested with neuraminidase (Clostridium perfringens, obtained from Pierce Chemical Co.) at 37 ° for 24 hours essentially according to the method of Cassidy et al. [J. T. Cassidy, G. W. Jourdian, and S. Roseman, J. Biol. Chem. 240, 3501 (1965)]. c The conditions used were 0.1 N H2S04, 80 °, 1 hour (R. G. Spiro, Vol. 8, p. 14). J See text for detail. The values reported were obtained by extrapolation of 16- and 32-hour values to zero time or infinite time, assuming first-order kinetics. • Obtained as a gift from Dr. E. A. Popenoe of Brookhaven National Laboratory. t Sheep submax mucin R-2 samples and its desialized product were obtained from Dr. S. Roseman of the Johns Hopkins University in 1962. a E. coli colominic acid was prepared from strain K235 by the method of Gotschlich. [E. C. Gotschlich, T. Y. Liu, and M. S. Artenstein, J. Exp. Med. 129, 1349 (1969)]. h T. Y. Liu, E. C. Gotschlich, F. T. Dunne, and E. K. Jonsson, J. Biol. Chem. 246, 4703 (1971). Obtained from Drs. Rebecca Lancefield and Emil C. Gotschlich of The Rockefeller University. w i t h 0.1 N H2S04 a t 80 ° for 1 hourlY; (2) t h e a c t i o n of n e u r a m i n i d a s e s ; a n d (3) a c i d - c a t a l y z e d m e t h a n o l y s i s w i t h a n h y d r o u s a c i d such as 2 N m e t h a n e s u l f o n i c acid. S i a l i c a c i d is in g e n e r a l u n s t a b l e in a q u e o u s acidic conditions. T h e s t a n d a r d m i l d c o n d i t i o n s (0.1 N H2SO4, 80 °, 1 hour) c o m m o n l y used a r e good p e r h a p s o n l y for t h e l i b e r a t i o n of sialic a c i d f r o m g l y c o p r o t e i n s b e c a u s e of its t e r m i n a l p o s i t i o n in these molecules. H o w e v e r , s t r o n g e r c o n d i t i o n s in a q u e o u s m e d i a will r a p i d l y cause c o m p l e t e d e s t r u c t i o n of s i a l i c acid. W h e n t h i s m e t h o d w a s a p p l i e d to p o l y m e r of sialic a c i d 1~R. G. Spiro, Vol. 8 [1].
[4]
DETERMINATION OF SIALIC ACID
53
with molecular weight in excess of 100,000 such as the meningococcal B- and C-polysaccharides and the E. coli colominic acid (see the table), the method failed to yield more than 20% of the sialic acid content in 60 minutes. Prolonged incubation (3-4 hours) resulted in higher recovery of sialic acid (30-50%), but the yield never exceeded 55% from both B- and C-polysaccharides of meningococcus.4 The failure of this procedure to yield more than 55% of the sialic acid from these polysaccharides is most likely caused by compensating factors; continuous release and destruction of the released sialic acid during the hydrolysis in 0.1 N H2S04 at 80 °. All or a large part of the sialic acid pre~ent in glycoproteins and polysaccharides can also usually be released by the action of neuraminidase (see the table). However, in some instances enzymatic hydrolysis of the sialic acid containing polysaccharide did not result in the release of any appreciable amount of sialic acid as are shown in the case of the C-polysaccharide from meningococcus4 and the polysaccharide isolated from a strain of group B streptococcus. Evidently, these neuraminidases are not capable of hydrolyzing certain glycosidic linkages of sialic acid or its O-acetylated derivatives. When 2 N methanesulfonic acid is used as a catalyst in methanolysis, the release of sialic acid from the polysaccharides or the glycoproteins has been consistently higher, as is shown in the table. For the estimation of sialic acid content in polysaccharide and glycoproteins, the advantages of using methanesulfonic acid as a catalyst for methanolysis, and the amino acid analyzer for the quantitative estimation of this amino sugar, are 3-fold. First, the reagent is effective in causing more complete cleavage of sialic acid from the polysaccharides and the glycoproteins. Second, the product of methanolysis after removal of solvent and saponification can be analyzed without further derivatization such as is required for the gas chromatographic procedure. Third, the product, methoxyneuraminic acid, is the most stable derivative of sialic acid and it is eluted at a unique position on the chromatogram of the amino acid analyzer column, which serves to identify this amino sugar. For the analysis of amino sugars in glycoproteins or polysaccharides, examination of the data as a function of hydrolysis time is important. It provides an opportunity to ascertain whether the particular amino sugar in question is stable under the condition of hydrolysis and if it has been completely liberated from the glycoproteins or the polysaccharides. It permits an extrapolation of the values, either to zero time or to infinite time to correct for the hydrolysis time on the destruction or release of amino sugars. This method of calculation prob-
54
ANALYTICXL METHODS
[S]
ably comes as close to giving accurate results as is possible at present. For this purpose at least two companion hydrolyzates, heated for 16 and 32 hours, are required. The values obtained at the two times of hydrolysis are extrapolated to give best figures.
[ 5 ] M o l e c u l a r W e i g h t D e t e r m i n a t i o n of G l y c o p r o t e i n s b y P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s in S o d i u m Dodecyl Sulfate B y JERE P. SEGREST a n d RICHARD L. JACKSON
Electrophoresis on polyacrylamide gels in the detergent sodium dodecyl sulfate [CH3(CH2)loCH2OSO3Na], abbreviated SDS, is a rapid and often employed technique for the determination of the molecular weights of proteins2 -3 The usefulness of this procedure for accurate molecular weight determinations depends upon two factors. (1) Proteins in general bind constant amounts of SDS per gram when saturated. 4,~ The protein then has an overall negative charge that masks its intrinsic charge, 2,3 resulting in a constant charge to mass ratio for proteins2 ,5 (2) Proteins saturated with SDS take on a rodlike configuration, the length of the structure being proportional to its polypeptide chain length, and thus its molecular weight? Principle This procedure is not directly applicable to molecular weight determinations of glycoproteins. Glycoproteins containing more than 1055 carbohydrate behave anomalously during SDS polyacrylamide gel electrophoresis when compared to standard proteins? ,r The cause of this anomalous behavior is a decreased binding of SDS per gram of glycoprotein as compared with standard proteins. 7 The lower SDS binding results in a decreased charge to mass ratio for glycoproteins versus I A. L. Shapiro, E. Vifiuela, and J. V. Maizel, Biochem. Biophys. Res. Commun. 28, 815 (1967). 2K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). s A. K. Danker and R. R. Rueekert, J. Biol. Chem. 244, 5074 (1969). "J. A. Reynolds and C. Tanford, Proc. Nat. Aead. Sei. U.8. 66, 1002 (1970). 5j. A. Reynolds and C. Tanford, J. Biol. Chem. 9,45, 5101 (1970). 8M. S. Bretscher, Nature (London) New Biol. 231, 229 (1971). J. P. Segrest, R. L. Jackson, E. P. Andrews, and V. T. Marehesi, Bioehem. Biophys. Res. Commun. 44, 390 (1971).