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Microdetermination of monosaccharides in glycoproteins
Microdetermination of Monosaccharides Glycoproteins. I. Electrophoretic
in
Methods’
INTRODUCTION Relatively large quantities of glycoproteins are required for carbohydrate analysis because of the limited sensitivity of current methods. The difficulty in obtaining large quantities of some biologically important glycoproteins such as cell surface components. viral glycoproteins, or components of complement has restricted research in this field. Limited information is available concerning carbohydrate composition and structure. and only rare examples of the specific role of complex carbohydrates in the biological function of these glycoproteins can be cited. The commonly used methods of carbohydrate analysis, such as gas chromatography (4, 10-12. l.5) or anion exchange chromatography in borate buffers (6). require the presence of at least 0. l- 1 pg of each monosaccharide in the sample. Recently reported was the construction of instruments. based on anion exchange chromatography. that increased the sensitivity of sugar detection. By using high-pressure columns with a cerate oxidimetric detector system (Y) or specially constructed detection units (a), as little as 1Om10mol of neutral sugars could be detected (8). Unfortunately most biochemical laboratories do not have access to such instruments. Howell et trl. (5) intr-educed a technique for determination of sugars in hydrolysates of glycoproteins that was based on labeling the monosaccharides with tritium by reduction with [3H]sodium borohydride. Alditols were separated by paper chromatography and sugar- concentr-ations were calculated from the tritium activity of the alditols. A modification of this technique was reported by Conrad et rrl. (3) who separated tritium-labeled alditols by ion exchange chromatography on filter paper and by Takasaki and Kobata (13) who for the same purpose used high voltage electrophoresis in borate buffers. ’ Supported by Grants CA-19918-01 and CA-19933 anal-dcd by the National tute, DHEW. 93
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The principles of the method reported here are similar to those of Takasaki and Kobata. A high degree of reproducibility was achieved by the addition of 14C-labeled monosaccharides of each sugar in the samples being analyzed. These served as internal standards that facilitated quantitation of the results. MATERIALS
S//grr,.s end reagents. o-Glucose and o-galactose were from Ffanstiehl Laboratories, Inc. (Waukegan, Ill): D-mannose, L-fucose, were from Calbiochem D-glucosamine-HCl, and D-ga]aCtOsamine-HCI (San Diego, Calif.), N-acetylneuraminic acid was from Sigma Chemical Company (St. Louis, MO.); o-[ I-‘4C]glucose, II-[ l-‘4C]galactose, and D-[l-14C]glucosamine-HCl of specific activity 60 mciimmole, n-[ l14C]mannose of specific activity 31.8 mCi/mmole, I.-[ I-14C]fucose of specific activity 61 mCi/mmole, and D-[ l-‘4C]galactosamine of specific activity 53 mCi/mmole were from AmershamiSearle Corporation (Arlington Heights, 111.):N-[4-‘4C]acetylneuraminic acid of specific activity 53.7 mCi/mmole, and [3H]sodium borohydride of specific activity 276 mCi/mmole were from New England Nuclear (Boston, Mass.). Ion exchange resins AG50W-X8 (200-400 mesh) in hydrogen form. AGI-X8 (200-400 mesh) in acetate form were from Bio-Rad Laboratories. Bu~@rs. Borate buffer 0.0157 M, pH 9.5, was prepared by dissolving 6 g of sodium tetraborate decahydrate in 500 ml water. adjusting the pH with 1.0 M NaOH and adding water to make a final volume of 1 liter. Borate buffers 0.15 M. pH 7.0-7.6 were prepared by dissolving 9.28 g of boric acid in 500 ml water, adjusting the desired pH with 1.0 M NaOH and adding water to make a final volume of 1 liter. Stun&wd solutiot~s of 14Cs~grr~.s.(1) A standard solution A, which was used for determination of neutral sugars and amino hexoses? was prepared by mixing [I-‘4C]labeled and unlabeled solutions of u-glucose, o-mannose, D-galactose, n-glucosamine-HCl, and n-galactosamine-HCl in such ratio that the specific activity of each sugar was 0.02 &i/nmole and the concentration of each sugar was 1 nmolelF1. (2) The standard solution B which was used for determination of sialic acids was prepared by mixing N-[4-14C]acetylneuraminic acid and unlabeled N-acetylneuraminic acid solutions to make a concentration of 1 nmoleipl with a specific activity of 0.01 &i/nmole. Glycopwteins. Secretory IgA and secretory components were isolated from human colostrum by Dr. Jiri Mestecky of the Institute of Dental z The term “neutral sugws” refers to n-glucose. o-mannose. “amino hexoses” refer to n-glucosamine and wgalactosamine
r)-galactose. throughout.
and I-fucose:
Research. School of Dentistry. University of Alabama at Birmingham. Methods of preparation of these proteins were described earlier (14). DESCRIPTION OF THE METHOD Srr/~z~>/e pwptrlvrfiotr. Glycoproteins before analysis were desalted by dialysis, gel filtration. or repeated precipitation of the protein with 90% ethanol. Glycoproteins not soluble in water were solubilized by incubation with pepsin. A water suspension of glycoprotein was acidified with diluted HCI to pH 1.5 and incubated with pepsin for several hours at 37°C at an enzyme-substrate ratio of 1:200. The glycoprotein sample that contained 40-100 pg of glycoproteins in a volume of -0.2 ml was usually divided into two aliquots. One aliquot was used for determination of sialic and neutral sugars. The second aliquot was used for determination of amino hexoses. Hydrolysis. Sialic acid w/asreleased by hydrolysis for 1 hr in 0.05 N HCI at 80°C. Neutral sugars were hydrolyzed 4 hr at 100°Cin 1 N HCI. Amino hexoses were liberated by hydrolysis at 100°Cin 1 N HCI for IO hr or in 3 N HCI for 4 hr. Septrrrrtiotr of’peptitlcs. The purpose of this step was to limit the consumption of [‘HIsodium borohydride for reduction of disulfide bonds and other functional groups in the polypeptide chain. Separation of peptides before the reduction of sugars with NaB’H, lowers the backround levels of radioactivity in the filter paper after electrophoresis allowing greater precision in the quantitation of sugars. The monosaccharides were separated from peptides and from excess HCI by ion exchange chromatography. The elution of monosaccharides from the ion exchange columns was monitored by measuring radioactivity of 14Cmonosaccharides in the standard solutions that were added to the hydrolysate. Five microliters each of standard solutions A and B (see Materials) was added to the aliquot of the sample that was designated fat determination of sialic acid and neutral sugars. Five microliters of standard solution A was added to the aliquot designated for determination of amino hexoses. The glass columns (0.7 cm in diameter) and pear-shaped lyophilization flasks used in the purification procedure were siliconized. (A) Sialic acid was separated from desialylated protein by passing the I-hr hydrolysate through a column containing 1 cm of anion exchange resin AG l-X8 (acetate). The fraction containing desialylated protein, which eluted from the column with distilled water. was collected, lyophilized, and subjected to further hydrolysis for determination of neutral sugars. Then a column of cation exchange resin AGSOW-XX(H-) was placed in tandem with the anion exchange column and the sialic acid was eluted with 0.25 M sodium acetate. (B) Neutral sugars released from the desialylated protein by hy-
drolysis in 1 N HCI for 4 hr were separated from peptides by ion exchange chromatography using two consecutive columns. The upper column contained 3 cm of AGI-X8 (acetate); the lower column, 3 cm of AG50W-X8 (H+). The hydrolysate before ion exchange chromatography was diluted with distilled water, to decrease the concentration of HCI to about 0.3 N. Neutral sugars were eluted with distilled water. Most of peptides and amino hexoses were retained in the lower, cation-exchange column. (C) The hydrolysate of glycoprotein designated for determination of amino hexoses was diluted with distilled water to reach the final HCI concentration of 0.3 N and applied to the same system of anion and cation exchange columns described for separation of neutral sugars. After the neutral sugars were eluted with distilled water the upper column was removed and the lower column was washed with 15 ml of 0.4 N pyridine. More than 90% of peptides and amino acids were removed in this step. The excess of pyridine was washed out of the column with 5 ml of distilled water. A column filled with 5 cm AC l-X8 (acetate) was placed in tandem with the cation exchange column and the amino hexoses were eluted with 1 N HCI. All sugar fractions after the column purification were lyophilized. Rcticlction c?f’slrg:rr~~.The residues of neutral sugars, amino hexoses. and sialic acids were redissolved in 100-200 ~1 of solution that contained 0.5- 1 mCi NaB’H, in 0.01 M NaOH. The specific activity of NaB’H, was approximately 300 mCi/mmole. The sample was reduced for 4 hr at 37°C. Reduction was terminated by adding 50 ~1 of glacial acetic acid, and the sample was freeze-dried. The residue was redissolved and lyophilized three more times with 100 ~1 of I .OM acetic acid. To remove borates, the residue was redissolved in 100 ~1 of methanol and evaporated under vacuum at room temperature. This treatment was reneated twice. Accurate monosaccharide determination is predicated on the quantitative reduction of all sugars. To determine the optimal excess of sodium borohydride required, under the conditions just described, r4C-labeled neutral sugars and amino hexoses were reduced with increasing amounts of [3Hlsodium borohydride. The ratio of 3H/14C increased with the increasing ratio of NaB’H,,‘carbohydrate and reached a maximal value when the sodium borohydride was present in an approximately 40 molar excess. The concentration of sodium borohydride in the reaction mixture also influences the quantitative reduction of monosaccharides. Using 14Clabeled sugars and a 50 molar excess of NaB”H,. it was determined that approximately 10 ~moleiml of sodium borohydride was the minimal concentration necessary for completing the reduction of neutral and amino sugars. Thus, using 1 mCi of NaB”H, of the specific activity -300 mCi/ mmole. the maximum volume of the reaction mixture that ensured complete reduction was -0.3 ml.
.\11~:ROI~EI~I~.R~IIN.~‘l-ION
01‘
~lONOS.-\(:(:l1.~RIDF.4
97
The kinetics of the reduction of Ri’acetylneuraminic acid is similar to that of neutral sugars and amino hexoses (2). Sodium borohydride reduces the carbonyl group on C-2. Under conditions used for reduction, a small amount of sialic acid appears to be decomposed. Since the sialic acid in the “C-labeled standard solution also undergo decomposition, these losses do not result in errors of quantitation. Elc(~r7.opl7o~r~si.s. The residues were dissolved in 50 ~1 of distilled water and 5-10 ,ul of the solution was applied to Whatman No. 3 filter paper (86 x 11 cm). Electrophoresis was performed at 2°C in a high-voltage electrophoretic system (Savant Instruments, Inc.) equipped with a cooling plate. Sialic acid was separated from contaminating compounds in borate buffer pH 7, (see Materials) at 48 V/cm for 1 hr. Neutral sugars were subjected to electrophoresis for 3 hr at 42 V/cm in borate buffer, pH 9.5. Amino hexoses were run for 3 hr at 48 V/cm in borate buffer. pH 7.6. Positions of the radioactive spots were determined by scanning for ‘“C-p emissitin on a chromatographic scanner (Packard, Model 7201). The paper strips were then cut into 0.5cm segments. The segments were dropped into scintillation vials and eluted for 2 hr with 0.5 ml distilled water. The ‘-‘C and ‘H activities were counted after addition of IO ml ACS scintillation cocktail (AmershamiSearle Corp., Arlington Heights. Ill.) in a scintillation counter (Nuclear Chigago. Model 730). Q7rtrr7tirofior7. The concentrations of sugars were computed from the difference in the ratio of ‘H:‘“C in the i4C-labeled standard solution and in a hydrolyzed glycoprotein sample to which the same 14C-labeled standard solution was added. The standard solution and sample were reduced and electrophoresed simultaneously under identical conditions. Accuracy of quantitation depends on the accuracy with which the concentrations of sugars in the standard solution were prepared. To obtain solutions of 14C-labeled sugars of desired concentration, the commercially supplied solutions of i4C sugars were diluted with solutions of nonlabeled sugars. The concentrations of i4C-labeled monosaccharides were calculated from manufacturers data. The freshly prepared standard solution was tested in the following way: Ten microliters of the standard solution (containing 10 nmoles of each i4C sugar) was reduced with [3H]sodium borohydride alone and also after addition of IO nmoles of unlabeled sugars. Both samples were subjected to electrophoretic separation. Computation of the results from r4C and jH activities pertaining to individual monosaccharides showed recovery of all sugars within the limit of + 5% indicating that manufacturer’s data on specific activities were accurate. The calculation of sugar concentrations from scintillation counter data was programmed for computation with a Sigma 7 computer. Lkscriprion c?f’tlre con~prrt~~~ pr~~grrr7~7. Two programs were required to obtain the final determination of sugar concentrations. The first program performs data correction for /3 energy spillover in the scintillation counter
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channels. The second program fits the corrected data to a sequence of normal distribution functions and calculates the concentrations of the unknown sugar samples. The data correction program calculates the efficiencies of counting iH and r4C in both scintillation counter channels and obtains the correct “H and r4C counts by solving the following two equations simultaneously for It and c: N;, = H,h + C,c + B,. N,, = H,,h + C,,c -c B,,,
where N = counts/min on a channel a or b, H.C = efficiencies of counting 3H and 14C, respectively, B = background counts for a given channel, h,c = disintegration per minute for 3H and r4C, respectively. The data correction program always expects two sets of input data during a run; the first corresponding to the standard sample, the second comesponding to the unknown sample. The program prints the input data and the corrected values for both sets of data. The corrected values are also stored on a disk for subsequent access by the fitting program. The curve fitting program performs a least-squares fit of the corrected data to the mathematical model.
The N exponential terms represent the normal-shaped curves corresponding to N peaks in the raw data. The two remaining terms represent a linear baseline. The nonlinear parameters h and a, are obtained using a Levenberg-Marquardt algorithm (7) and the linear parameters are obtained using conventional multiple linear regression. After finding the best fit, integrals are calculated for each peak as follows:
The concentration of an unknown sugar is calculated in the following manner:
where r/, = concentration of unknown for the 17thpeak (np), S, = concentration of standard for the /rth peak (UP), Th,n = area of 3H peak of unknown,
99 TC,,!= area of 14C peak of unknown, TL,,,= area of jH peak of standard, Ti, = area of 14C peak of standard. F(&res 1 and 2 are computer records showing separation and integration of sugar alditols from human colostral IgA immunoglobulin. The method of least-squares facilitates quantitation of the sugars even when they were not perfectly separated. The use of ‘“C-labeled sugars as an internal standard allows compensation for physical and chemical losses resulting from manipulation of the sample. Table I compares the sugar
SECRETORY
IMMUNOGLOBULIN 6 : 5 81
A
L : I d
FIL. 1. Computer record representing separation of neutral sugars from s-IgA. Forty-five micrograms of s-IgA was hydrolyzed in I hi HCI for 4 hr at 100°C. Five nanomoles each of I>-[ I-Y]glucose. o-[l-14C]mannose. and II-[ I-“Clgalactose was added as an internal standard. Peptides were separated by ion exchange chromatography. Monosaccharides were reduced with 1 mCi of NaBJH, (270 mCi/mmole) and electrophoresed as described in the text. Peaks from the left: (I) L)-glucose. (2) n-mannose and I-fucose. and (3) u-galactose. The two upper graphs represent crude data from the computer: left. ‘H: right. “C radioactivity. The two lower graphs represent data corrected by the least-squares method.
100
I‘OMASA
SECRETORY
ET AL lMMUNOCLO8ULlN “0 7 8
A
Frc. 2. Computer record representing separation of amino hexoses. Amino hexoses were determined in the same hydrolysate of s-IgA as neutral sugars. internal standard contained 5 mmol of I>-[ I-‘4C]glucosamine-HCI and u-[ l-14C]galactosamine-HCI. Left peaks represent I)-glucosamine: right peaks u-galactosamine. The two upper graphs represent crude data The two lower graphs represent data from the computer: left. JH: right, “Y radioactivity. corrected
by the least-squares
method.
analyses of human secretory IgA using gas liquid chromatography, colorimetry, and radioelectrophoretic method. Precision of the mdioelect~opfioueti~. rn~t~~t~i~. Precision was determined by analyzing a series of six 50-pg samples of secretory component isolated from human colostrum. The protein was hydrolyzed in 0.05 M HCl for I hr at 80°C to determine sialic acid content and in 1 N HCl for 4 hr at 100°C to assay neutral sugars and amino hexoses. The analyses showed the following results (expressed in nanomolesE0 ~g protein 4 standard deviation):glucosamine (free base) 17.92 k 0.72; mannose + fucose 21.29 rt 0.73: galactose 9.75 i 0.27; sialic acid 2.22 2 0.07. The coefficients of variation were in the range 2.8-4.0%. For comparison:
TABLE
GLC of alditol acetates (III)
I
Thiobarbituric acid assay (I )
Radioelectrophoresis
Amount of protein analyzed (pg) Carbohydrate
1900
Fucose Mannose Galactose Glucose Glucosamine Galactosamine Sialic Acid
0.94 1.83 I.85 0.17 3.30 0.38
Total
1200 I.8 II.27
95 4.04 I.78 0.17 3.6X 0.35 I.75 II.77
(I Gram/ 100 grams glycoprotein
The coefficients of variation for determination of neutral and amino sugars in 1.2 mg of human secretory IgA by gas chromatography of alditol acetates were in the range of 58%. DISCUSSION
Separation of tritium-labeled sugar alditols and reduced sialic acids by high-voltage electrophoresis on filter paper and quantitation of sugars by measuring the incorporated radioactivity was previously reported by Takasaki and Kobata (13). Our results differ in several ways from theirs: (a) In borate buffers, pH 9.5. under conditions described by Takasaki and Kobata, we were unable to achieve electrophoretic separation of fucitol from mannitol or of glucosaminitol from galactosaminitol. (b) The condition of hydrolysis described by those investigators for determination of monosaccharides in “complex sugar samples” did not give satisfactory results when applied in our laboratory for determination of sugars in glycoproteins. (c) The procedure described (13) does not include any purification steps for removing excess salt or peptides prior to performing the electrophoretic analysis. When peptides are present, part of the [3H]sodium borohydride is consumed in reducing them with the result that the sugars may be incompletely reduced. Furthermore, the presence of peptides and large amounts of salts in the sample interfere with electrophoretic separation, resulting in errors in quantitation. (d) We found that the use of internal standards was essential for obtaining reliable results. Howell et crl. (5) used chromatographic techniques to separate and
102
TOMANA
ET AL
quantitate tritium-labeled alditols of sugars. In our experience chromatography on filter paper results in reasonable separation of alditols from pure sugar mixtures. The presence of salts and peptides in glycoprotein hydrolysates usually causes less sharp separation and consequently less precise quantitation of sugars. The method described is highly sensitive and reproducible. The sensitivity depends, to a certain degree, on the specific activity of [3H]sodium borohydride and on the specific activity and quantity of i4C sugars in the internal standard. In our analyses we used sodium borohydride with a specific activity of approximately 300 mCi/mmol. The internal standard contained l-5 nanomoles of i4C-labeled monosaccharides of the specific activity 2 &i/100 nmole. Under these conditions we were able to determine the carbohydrate content in 50- 100 pg of glycoproteins containing 6-207~ carbohydrates. The coefficient of variation in determination of monosaccharides in 50 pugof a glycoprotein (secretory component) that contained about 17% of sugars was in the range of 2.8-4.0s. The high degree of accuracy and reliability achieved by the present method is attributed to the inclusion of an internal standard consisting of i4C-labeled sugars of all the monosaccharides in the sample. This technique assures compensation for the chemical or the physical losses incurred in the course of the analytical procedure. The chief disadvantage of the method described in this paper is its failure to allow separate quantitation of fucose and mannose. Both these sugars in their reduced form could be separated by chromatography on ion-exchange papers (3). Studies are presently in progress in our laboratory to establish the reproducibility of determination of monosaccharides in glycoproteins using a combination of electrophoresis and ion exchange chromatography. SUMMARY A sensitive method that has been developed for determination of monosaccharides in glycoproteins is based on high-voltage electrophoretic separation of tritium-labeled monosaccharides. Precise quantitation is mediated by using internal standards. specific for each monosaccharide in the sample. The application of this method and its reproducibility has been demonstrated by analysis of the carbohydrate moiety of immunoglobulins.
ACKNOWLEDGMENTS The authors wish to thank Dr. Jiri Dubovsky for editorial advice.
for helpful discussions and Mrs. C. A. Sims
REFERENCES I. Aminoff. D.. Methods for the estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Bioc,/,c,r?r. J. 81, 384-392 (1962). 2. Blix, G., Lindberg, E.. Odin. L., and Werner, I., Studies on sialic acids. Ac,ttr. Sot. Med. Upstrl. 61, l-25 (1956). 3. Conrad. H. E.. Varboncouer. E.. and James, M. E.. Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion-exchange papers. A/?tr/. Bk~hrr,~. 51, 486-500 ( 1973).
MICRODET
ERhlIN.-\TION
OF MOSOS-\CCH~RIDES
103
4.
Eisenherg, F.. Jr., Cyclic butaneboronic acid esters: novel derivatives for the rapid separation of carbohydrates by gas-liquid chromatography. C‘rrrhoh~tl. KU. 19, 135-138 (1971). 5. Howell, H. M.. Conrad. H. E., and Voss. E. W.. Jr.. Radiochromatographic carbohydrate analysis and its application to gamma globulin systems. Fed. Ptw. 30, 594 (1971). 6. Lee. Y. C.. McKelvy. J. R.. and Lang. D.. Rapid automatic analysis of sugar components of glycoproteins. Il. Neutral sugars. A~rrl. H~~~(~/I(,Iu. 27, 567-574 (1969). 7. Marquardt, D. W.. An algorithm for least squares estimation of nonlinear parameters.J. Sot.. Iutl. A/@. Morlr. 2, 43 I-441 ( 1963). H. Morrison. W. H.. Lou, M. F.. and Hamilton. P. B.. The determination of hexoses and pentoses by anion-exchange chromatography: A method of high sensitivity. Autrl. Bioc~/lc~m. 71, 414-3’5 (19761.
Y. Mrochek. J. E.. Dinsmore, S. R.. and Waalkes. T. P.. Liquid-chromatographic analysis for neutral carbohydrates in serum glycoproteins. C/i/l. C‘/ICIU. 21, 1314- 1322 (1975). 10. Niedermeier. W.. Gas chromatography of neutral and amino sugars in glycoproteins. Autrl. Bioc~hc,/,~. 40, 465-475 ( 1971). II. Sawardeker. J. S.. Sloneker. J. H.. and Jeanes. A., Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Autrl. Chc~n~. 37, 160?- 1604 (1965). 12. Sweeley. C. C.. Bentley. E.. Makita. M. and Wells. W. W.. Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. ./. Amer. Chc~m. SOCK. 85, 2497-2507 (1963). 13. Takasaki. S.. and Kobata, A. J.. Microdetermination of individual neutral and amino sugars and Ri-acetylneuraminic acid in complex saccharides. ./. Bioc,llc~/?~. (Tokyo) 76, 783-788 (1974). W.. Studies on human secretory 14. Tomana. M.. Mestecky, J., and Niedermeier, immunoglobulin A. IV. Carbohydrate composition. .I. /rrl,~r,~o/. 108, I631 ~ 1636 (1972). 1.5. Zaneta, J. P.. Breckenridge. W. C.. and Wincendon. G.. Analysis of monosaccharides by liqud chromatography of the O-methyl glycosides as trifluoroacetate derivatives. J. C/I,-omtrto~v. 69, 291 -303 ( 1972).
in
Methods’
INTRODUCTION Relatively large quantities of glycoproteins are required for carbohydrate analysis because of the limited sensitivity of current methods. The difficulty in obtaining large quantities of some biologically important glycoproteins such as cell surface components. viral glycoproteins, or components of complement has restricted research in this field. Limited information is available concerning carbohydrate composition and structure. and only rare examples of the specific role of complex carbohydrates in the biological function of these glycoproteins can be cited. The commonly used methods of carbohydrate analysis, such as gas chromatography (4, 10-12. l.5) or anion exchange chromatography in borate buffers (6). require the presence of at least 0. l- 1 pg of each monosaccharide in the sample. Recently reported was the construction of instruments. based on anion exchange chromatography. that increased the sensitivity of sugar detection. By using high-pressure columns with a cerate oxidimetric detector system (Y) or specially constructed detection units (a), as little as 1Om10mol of neutral sugars could be detected (8). Unfortunately most biochemical laboratories do not have access to such instruments. Howell et trl. (5) intr-educed a technique for determination of sugars in hydrolysates of glycoproteins that was based on labeling the monosaccharides with tritium by reduction with [3H]sodium borohydride. Alditols were separated by paper chromatography and sugar- concentr-ations were calculated from the tritium activity of the alditols. A modification of this technique was reported by Conrad et rrl. (3) who separated tritium-labeled alditols by ion exchange chromatography on filter paper and by Takasaki and Kobata (13) who for the same purpose used high voltage electrophoresis in borate buffers. ’ Supported by Grants CA-19918-01 and CA-19933 anal-dcd by the National tute, DHEW. 93
Cancel- Insti-
94
TOMAKA
ET AL
The principles of the method reported here are similar to those of Takasaki and Kobata. A high degree of reproducibility was achieved by the addition of 14C-labeled monosaccharides of each sugar in the samples being analyzed. These served as internal standards that facilitated quantitation of the results. MATERIALS
S//grr,.s end reagents. o-Glucose and o-galactose were from Ffanstiehl Laboratories, Inc. (Waukegan, Ill): D-mannose, L-fucose, were from Calbiochem D-glucosamine-HCl, and D-ga]aCtOsamine-HCI (San Diego, Calif.), N-acetylneuraminic acid was from Sigma Chemical Company (St. Louis, MO.); o-[ I-‘4C]glucose, II-[ l-‘4C]galactose, and D-[l-14C]glucosamine-HCl of specific activity 60 mciimmole, n-[ l14C]mannose of specific activity 31.8 mCi/mmole, I.-[ I-14C]fucose of specific activity 61 mCi/mmole, and D-[ l-‘4C]galactosamine of specific activity 53 mCi/mmole were from AmershamiSearle Corporation (Arlington Heights, 111.):N-[4-‘4C]acetylneuraminic acid of specific activity 53.7 mCi/mmole, and [3H]sodium borohydride of specific activity 276 mCi/mmole were from New England Nuclear (Boston, Mass.). Ion exchange resins AG50W-X8 (200-400 mesh) in hydrogen form. AGI-X8 (200-400 mesh) in acetate form were from Bio-Rad Laboratories. Bu~@rs. Borate buffer 0.0157 M, pH 9.5, was prepared by dissolving 6 g of sodium tetraborate decahydrate in 500 ml water. adjusting the pH with 1.0 M NaOH and adding water to make a final volume of 1 liter. Borate buffers 0.15 M. pH 7.0-7.6 were prepared by dissolving 9.28 g of boric acid in 500 ml water, adjusting the desired pH with 1.0 M NaOH and adding water to make a final volume of 1 liter. Stun&wd solutiot~s of 14Cs~grr~.s.(1) A standard solution A, which was used for determination of neutral sugars and amino hexoses? was prepared by mixing [I-‘4C]labeled and unlabeled solutions of u-glucose, o-mannose, D-galactose, n-glucosamine-HCl, and n-galactosamine-HCl in such ratio that the specific activity of each sugar was 0.02 &i/nmole and the concentration of each sugar was 1 nmolelF1. (2) The standard solution B which was used for determination of sialic acids was prepared by mixing N-[4-14C]acetylneuraminic acid and unlabeled N-acetylneuraminic acid solutions to make a concentration of 1 nmoleipl with a specific activity of 0.01 &i/nmole. Glycopwteins. Secretory IgA and secretory components were isolated from human colostrum by Dr. Jiri Mestecky of the Institute of Dental z The term “neutral sugws” refers to n-glucose. o-mannose. “amino hexoses” refer to n-glucosamine and wgalactosamine
r)-galactose. throughout.
and I-fucose:
Research. School of Dentistry. University of Alabama at Birmingham. Methods of preparation of these proteins were described earlier (14). DESCRIPTION OF THE METHOD Srr/~z~>/e pwptrlvrfiotr. Glycoproteins before analysis were desalted by dialysis, gel filtration. or repeated precipitation of the protein with 90% ethanol. Glycoproteins not soluble in water were solubilized by incubation with pepsin. A water suspension of glycoprotein was acidified with diluted HCI to pH 1.5 and incubated with pepsin for several hours at 37°C at an enzyme-substrate ratio of 1:200. The glycoprotein sample that contained 40-100 pg of glycoproteins in a volume of -0.2 ml was usually divided into two aliquots. One aliquot was used for determination of sialic and neutral sugars. The second aliquot was used for determination of amino hexoses. Hydrolysis. Sialic acid w/asreleased by hydrolysis for 1 hr in 0.05 N HCI at 80°C. Neutral sugars were hydrolyzed 4 hr at 100°Cin 1 N HCI. Amino hexoses were liberated by hydrolysis at 100°Cin 1 N HCI for IO hr or in 3 N HCI for 4 hr. Septrrrrtiotr of’peptitlcs. The purpose of this step was to limit the consumption of [‘HIsodium borohydride for reduction of disulfide bonds and other functional groups in the polypeptide chain. Separation of peptides before the reduction of sugars with NaB’H, lowers the backround levels of radioactivity in the filter paper after electrophoresis allowing greater precision in the quantitation of sugars. The monosaccharides were separated from peptides and from excess HCI by ion exchange chromatography. The elution of monosaccharides from the ion exchange columns was monitored by measuring radioactivity of 14Cmonosaccharides in the standard solutions that were added to the hydrolysate. Five microliters each of standard solutions A and B (see Materials) was added to the aliquot of the sample that was designated fat determination of sialic acid and neutral sugars. Five microliters of standard solution A was added to the aliquot designated for determination of amino hexoses. The glass columns (0.7 cm in diameter) and pear-shaped lyophilization flasks used in the purification procedure were siliconized. (A) Sialic acid was separated from desialylated protein by passing the I-hr hydrolysate through a column containing 1 cm of anion exchange resin AG l-X8 (acetate). The fraction containing desialylated protein, which eluted from the column with distilled water. was collected, lyophilized, and subjected to further hydrolysis for determination of neutral sugars. Then a column of cation exchange resin AGSOW-XX(H-) was placed in tandem with the anion exchange column and the sialic acid was eluted with 0.25 M sodium acetate. (B) Neutral sugars released from the desialylated protein by hy-
drolysis in 1 N HCI for 4 hr were separated from peptides by ion exchange chromatography using two consecutive columns. The upper column contained 3 cm of AGI-X8 (acetate); the lower column, 3 cm of AG50W-X8 (H+). The hydrolysate before ion exchange chromatography was diluted with distilled water, to decrease the concentration of HCI to about 0.3 N. Neutral sugars were eluted with distilled water. Most of peptides and amino hexoses were retained in the lower, cation-exchange column. (C) The hydrolysate of glycoprotein designated for determination of amino hexoses was diluted with distilled water to reach the final HCI concentration of 0.3 N and applied to the same system of anion and cation exchange columns described for separation of neutral sugars. After the neutral sugars were eluted with distilled water the upper column was removed and the lower column was washed with 15 ml of 0.4 N pyridine. More than 90% of peptides and amino acids were removed in this step. The excess of pyridine was washed out of the column with 5 ml of distilled water. A column filled with 5 cm AC l-X8 (acetate) was placed in tandem with the cation exchange column and the amino hexoses were eluted with 1 N HCI. All sugar fractions after the column purification were lyophilized. Rcticlction c?f’slrg:rr~~.The residues of neutral sugars, amino hexoses. and sialic acids were redissolved in 100-200 ~1 of solution that contained 0.5- 1 mCi NaB’H, in 0.01 M NaOH. The specific activity of NaB’H, was approximately 300 mCi/mmole. The sample was reduced for 4 hr at 37°C. Reduction was terminated by adding 50 ~1 of glacial acetic acid, and the sample was freeze-dried. The residue was redissolved and lyophilized three more times with 100 ~1 of I .OM acetic acid. To remove borates, the residue was redissolved in 100 ~1 of methanol and evaporated under vacuum at room temperature. This treatment was reneated twice. Accurate monosaccharide determination is predicated on the quantitative reduction of all sugars. To determine the optimal excess of sodium borohydride required, under the conditions just described, r4C-labeled neutral sugars and amino hexoses were reduced with increasing amounts of [3Hlsodium borohydride. The ratio of 3H/14C increased with the increasing ratio of NaB’H,,‘carbohydrate and reached a maximal value when the sodium borohydride was present in an approximately 40 molar excess. The concentration of sodium borohydride in the reaction mixture also influences the quantitative reduction of monosaccharides. Using 14Clabeled sugars and a 50 molar excess of NaB”H,. it was determined that approximately 10 ~moleiml of sodium borohydride was the minimal concentration necessary for completing the reduction of neutral and amino sugars. Thus, using 1 mCi of NaB”H, of the specific activity -300 mCi/ mmole. the maximum volume of the reaction mixture that ensured complete reduction was -0.3 ml.
.\11~:ROI~EI~I~.R~IIN.~‘l-ION
01‘
~lONOS.-\(:(:l1.~RIDF.4
97
The kinetics of the reduction of Ri’acetylneuraminic acid is similar to that of neutral sugars and amino hexoses (2). Sodium borohydride reduces the carbonyl group on C-2. Under conditions used for reduction, a small amount of sialic acid appears to be decomposed. Since the sialic acid in the “C-labeled standard solution also undergo decomposition, these losses do not result in errors of quantitation. Elc(~r7.opl7o~r~si.s. The residues were dissolved in 50 ~1 of distilled water and 5-10 ,ul of the solution was applied to Whatman No. 3 filter paper (86 x 11 cm). Electrophoresis was performed at 2°C in a high-voltage electrophoretic system (Savant Instruments, Inc.) equipped with a cooling plate. Sialic acid was separated from contaminating compounds in borate buffer pH 7, (see Materials) at 48 V/cm for 1 hr. Neutral sugars were subjected to electrophoresis for 3 hr at 42 V/cm in borate buffer, pH 9.5. Amino hexoses were run for 3 hr at 48 V/cm in borate buffer. pH 7.6. Positions of the radioactive spots were determined by scanning for ‘“C-p emissitin on a chromatographic scanner (Packard, Model 7201). The paper strips were then cut into 0.5cm segments. The segments were dropped into scintillation vials and eluted for 2 hr with 0.5 ml distilled water. The ‘-‘C and ‘H activities were counted after addition of IO ml ACS scintillation cocktail (AmershamiSearle Corp., Arlington Heights. Ill.) in a scintillation counter (Nuclear Chigago. Model 730). Q7rtrr7tirofior7. The concentrations of sugars were computed from the difference in the ratio of ‘H:‘“C in the i4C-labeled standard solution and in a hydrolyzed glycoprotein sample to which the same 14C-labeled standard solution was added. The standard solution and sample were reduced and electrophoresed simultaneously under identical conditions. Accuracy of quantitation depends on the accuracy with which the concentrations of sugars in the standard solution were prepared. To obtain solutions of 14C-labeled sugars of desired concentration, the commercially supplied solutions of i4C sugars were diluted with solutions of nonlabeled sugars. The concentrations of i4C-labeled monosaccharides were calculated from manufacturers data. The freshly prepared standard solution was tested in the following way: Ten microliters of the standard solution (containing 10 nmoles of each i4C sugar) was reduced with [3H]sodium borohydride alone and also after addition of IO nmoles of unlabeled sugars. Both samples were subjected to electrophoretic separation. Computation of the results from r4C and jH activities pertaining to individual monosaccharides showed recovery of all sugars within the limit of + 5% indicating that manufacturer’s data on specific activities were accurate. The calculation of sugar concentrations from scintillation counter data was programmed for computation with a Sigma 7 computer. Lkscriprion c?f’tlre con~prrt~~~ pr~~grrr7~7. Two programs were required to obtain the final determination of sugar concentrations. The first program performs data correction for /3 energy spillover in the scintillation counter
98
TOMAN.
ET AL
channels. The second program fits the corrected data to a sequence of normal distribution functions and calculates the concentrations of the unknown sugar samples. The data correction program calculates the efficiencies of counting iH and r4C in both scintillation counter channels and obtains the correct “H and r4C counts by solving the following two equations simultaneously for It and c: N;, = H,h + C,c + B,. N,, = H,,h + C,,c -c B,,,
where N = counts/min on a channel a or b, H.C = efficiencies of counting 3H and 14C, respectively, B = background counts for a given channel, h,c = disintegration per minute for 3H and r4C, respectively. The data correction program always expects two sets of input data during a run; the first corresponding to the standard sample, the second comesponding to the unknown sample. The program prints the input data and the corrected values for both sets of data. The corrected values are also stored on a disk for subsequent access by the fitting program. The curve fitting program performs a least-squares fit of the corrected data to the mathematical model.
The N exponential terms represent the normal-shaped curves corresponding to N peaks in the raw data. The two remaining terms represent a linear baseline. The nonlinear parameters h and a, are obtained using a Levenberg-Marquardt algorithm (7) and the linear parameters are obtained using conventional multiple linear regression. After finding the best fit, integrals are calculated for each peak as follows:
The concentration of an unknown sugar is calculated in the following manner:
where r/, = concentration of unknown for the 17thpeak (np), S, = concentration of standard for the /rth peak (UP), Th,n = area of 3H peak of unknown,
99 TC,,!= area of 14C peak of unknown, TL,,,= area of jH peak of standard, Ti, = area of 14C peak of standard. F(&res 1 and 2 are computer records showing separation and integration of sugar alditols from human colostral IgA immunoglobulin. The method of least-squares facilitates quantitation of the sugars even when they were not perfectly separated. The use of ‘“C-labeled sugars as an internal standard allows compensation for physical and chemical losses resulting from manipulation of the sample. Table I compares the sugar
SECRETORY
IMMUNOGLOBULIN 6 : 5 81
A
L : I d
FIL. 1. Computer record representing separation of neutral sugars from s-IgA. Forty-five micrograms of s-IgA was hydrolyzed in I hi HCI for 4 hr at 100°C. Five nanomoles each of I>-[ I-Y]glucose. o-[l-14C]mannose. and II-[ I-“Clgalactose was added as an internal standard. Peptides were separated by ion exchange chromatography. Monosaccharides were reduced with 1 mCi of NaBJH, (270 mCi/mmole) and electrophoresed as described in the text. Peaks from the left: (I) L)-glucose. (2) n-mannose and I-fucose. and (3) u-galactose. The two upper graphs represent crude data from the computer: left. ‘H: right. “C radioactivity. The two lower graphs represent data corrected by the least-squares method.
100
I‘OMASA
SECRETORY
ET AL lMMUNOCLO8ULlN “0 7 8
A
Frc. 2. Computer record representing separation of amino hexoses. Amino hexoses were determined in the same hydrolysate of s-IgA as neutral sugars. internal standard contained 5 mmol of I>-[ I-‘4C]glucosamine-HCI and u-[ l-14C]galactosamine-HCI. Left peaks represent I)-glucosamine: right peaks u-galactosamine. The two upper graphs represent crude data The two lower graphs represent data from the computer: left. JH: right, “Y radioactivity. corrected
by the least-squares
method.
analyses of human secretory IgA using gas liquid chromatography, colorimetry, and radioelectrophoretic method. Precision of the mdioelect~opfioueti~. rn~t~~t~i~. Precision was determined by analyzing a series of six 50-pg samples of secretory component isolated from human colostrum. The protein was hydrolyzed in 0.05 M HCl for I hr at 80°C to determine sialic acid content and in 1 N HCl for 4 hr at 100°C to assay neutral sugars and amino hexoses. The analyses showed the following results (expressed in nanomolesE0 ~g protein 4 standard deviation):glucosamine (free base) 17.92 k 0.72; mannose + fucose 21.29 rt 0.73: galactose 9.75 i 0.27; sialic acid 2.22 2 0.07. The coefficients of variation were in the range 2.8-4.0%. For comparison:
TABLE
GLC of alditol acetates (III)
I
Thiobarbituric acid assay (I )
Radioelectrophoresis
Amount of protein analyzed (pg) Carbohydrate
1900
Fucose Mannose Galactose Glucose Glucosamine Galactosamine Sialic Acid
0.94 1.83 I.85 0.17 3.30 0.38
Total
1200 I.8 II.27
95 4.04 I.78 0.17 3.6X 0.35 I.75 II.77
(I Gram/ 100 grams glycoprotein
The coefficients of variation for determination of neutral and amino sugars in 1.2 mg of human secretory IgA by gas chromatography of alditol acetates were in the range of 58%. DISCUSSION
Separation of tritium-labeled sugar alditols and reduced sialic acids by high-voltage electrophoresis on filter paper and quantitation of sugars by measuring the incorporated radioactivity was previously reported by Takasaki and Kobata (13). Our results differ in several ways from theirs: (a) In borate buffers, pH 9.5. under conditions described by Takasaki and Kobata, we were unable to achieve electrophoretic separation of fucitol from mannitol or of glucosaminitol from galactosaminitol. (b) The condition of hydrolysis described by those investigators for determination of monosaccharides in “complex sugar samples” did not give satisfactory results when applied in our laboratory for determination of sugars in glycoproteins. (c) The procedure described (13) does not include any purification steps for removing excess salt or peptides prior to performing the electrophoretic analysis. When peptides are present, part of the [3H]sodium borohydride is consumed in reducing them with the result that the sugars may be incompletely reduced. Furthermore, the presence of peptides and large amounts of salts in the sample interfere with electrophoretic separation, resulting in errors in quantitation. (d) We found that the use of internal standards was essential for obtaining reliable results. Howell et crl. (5) used chromatographic techniques to separate and
102
TOMANA
ET AL
quantitate tritium-labeled alditols of sugars. In our experience chromatography on filter paper results in reasonable separation of alditols from pure sugar mixtures. The presence of salts and peptides in glycoprotein hydrolysates usually causes less sharp separation and consequently less precise quantitation of sugars. The method described is highly sensitive and reproducible. The sensitivity depends, to a certain degree, on the specific activity of [3H]sodium borohydride and on the specific activity and quantity of i4C sugars in the internal standard. In our analyses we used sodium borohydride with a specific activity of approximately 300 mCi/mmol. The internal standard contained l-5 nanomoles of i4C-labeled monosaccharides of the specific activity 2 &i/100 nmole. Under these conditions we were able to determine the carbohydrate content in 50- 100 pg of glycoproteins containing 6-207~ carbohydrates. The coefficient of variation in determination of monosaccharides in 50 pugof a glycoprotein (secretory component) that contained about 17% of sugars was in the range of 2.8-4.0s. The high degree of accuracy and reliability achieved by the present method is attributed to the inclusion of an internal standard consisting of i4C-labeled sugars of all the monosaccharides in the sample. This technique assures compensation for the chemical or the physical losses incurred in the course of the analytical procedure. The chief disadvantage of the method described in this paper is its failure to allow separate quantitation of fucose and mannose. Both these sugars in their reduced form could be separated by chromatography on ion-exchange papers (3). Studies are presently in progress in our laboratory to establish the reproducibility of determination of monosaccharides in glycoproteins using a combination of electrophoresis and ion exchange chromatography. SUMMARY A sensitive method that has been developed for determination of monosaccharides in glycoproteins is based on high-voltage electrophoretic separation of tritium-labeled monosaccharides. Precise quantitation is mediated by using internal standards. specific for each monosaccharide in the sample. The application of this method and its reproducibility has been demonstrated by analysis of the carbohydrate moiety of immunoglobulins.
ACKNOWLEDGMENTS The authors wish to thank Dr. Jiri Dubovsky for editorial advice.
for helpful discussions and Mrs. C. A. Sims
REFERENCES I. Aminoff. D.. Methods for the estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Bioc,/,c,r?r. J. 81, 384-392 (1962). 2. Blix, G., Lindberg, E.. Odin. L., and Werner, I., Studies on sialic acids. Ac,ttr. Sot. Med. Upstrl. 61, l-25 (1956). 3. Conrad. H. E.. Varboncouer. E.. and James, M. E.. Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion-exchange papers. A/?tr/. Bk~hrr,~. 51, 486-500 ( 1973).
MICRODET
ERhlIN.-\TION
OF MOSOS-\CCH~RIDES
103
4.
Eisenherg, F.. Jr., Cyclic butaneboronic acid esters: novel derivatives for the rapid separation of carbohydrates by gas-liquid chromatography. C‘rrrhoh~tl. KU. 19, 135-138 (1971). 5. Howell, H. M.. Conrad. H. E., and Voss. E. W.. Jr.. Radiochromatographic carbohydrate analysis and its application to gamma globulin systems. Fed. Ptw. 30, 594 (1971). 6. Lee. Y. C.. McKelvy. J. R.. and Lang. D.. Rapid automatic analysis of sugar components of glycoproteins. Il. Neutral sugars. A~rrl. H~~~(~/I(,Iu. 27, 567-574 (1969). 7. Marquardt, D. W.. An algorithm for least squares estimation of nonlinear parameters.J. Sot.. Iutl. A/@. Morlr. 2, 43 I-441 ( 1963). H. Morrison. W. H.. Lou, M. F.. and Hamilton. P. B.. The determination of hexoses and pentoses by anion-exchange chromatography: A method of high sensitivity. Autrl. Bioc~/lc~m. 71, 414-3’5 (19761.
Y. Mrochek. J. E.. Dinsmore, S. R.. and Waalkes. T. P.. Liquid-chromatographic analysis for neutral carbohydrates in serum glycoproteins. C/i/l. C‘/ICIU. 21, 1314- 1322 (1975). 10. Niedermeier. W.. Gas chromatography of neutral and amino sugars in glycoproteins. Autrl. Bioc~hc,/,~. 40, 465-475 ( 1971). II. Sawardeker. J. S.. Sloneker. J. H.. and Jeanes. A., Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Autrl. Chc~n~. 37, 160?- 1604 (1965). 12. Sweeley. C. C.. Bentley. E.. Makita. M. and Wells. W. W.. Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. ./. Amer. Chc~m. SOCK. 85, 2497-2507 (1963). 13. Takasaki. S.. and Kobata, A. J.. Microdetermination of individual neutral and amino sugars and Ri-acetylneuraminic acid in complex saccharides. ./. Bioc,llc~/?~. (Tokyo) 76, 783-788 (1974). W.. Studies on human secretory 14. Tomana. M.. Mestecky, J., and Niedermeier, immunoglobulin A. IV. Carbohydrate composition. .I. /rrl,~r,~o/. 108, I631 ~ 1636 (1972). 1.5. Zaneta, J. P.. Breckenridge. W. C.. and Wincendon. G.. Analysis of monosaccharides by liqud chromatography of the O-methyl glycosides as trifluoroacetate derivatives. J. C/I,-omtrto~v. 69, 291 -303 ( 1972).