ANALYTICAL

BIOCHEMISTRY

Procedures

84,

539-550 (1978)

for the Automated Analyses and Glycosaminoglycans

of Proteoglycans

J. D. FORD AND J. R. BAKER The

Institute

of Dental Research, School of Dentistry and University of Alabama in Birmingham, Birmingham,

Department of Medicine, Alabama 35294

Received May 24, 1977; accepted September 19, 1977 Methods for the automated analysis of hexose, uranic acid, and protein using the Technicon AutoAnalyzer II have been developed by modifying previously published procedures. A method of separating glucosamine and galactosamine, which is eminently suited to quantitating one in the presence of alarge amount ofthe other, is reported, Procedures that can be recommended for determining the amino acid content and individual neutral sugars of proteoglycans or glycosaminoglycans are also described.

Chemical analysis of proteoglycans involves separate estimations of amino acids, hexosamines, neutral sugars, uranic acids, and sulfate. There are some problems peculiar to proteoglycans in determining certain classes of these compounds. For example, after acid hydrolysis of the sample, amino acids must be completely resolved from a relatively much larger amount of hexosamine. Some procedures (l-3), based upon the methods of Spackman et al. (4), have been designed to overcome this difficulty. Neutral sugars may be determined by ion exhange chromatography of their borate complexes as originally described by Khym and Zill (5). A number of methods for the determination of neutral sugars in glycoproteins have been developed (6-lo), which are based upon this system. For the multisample determination of uranic acid, hexose, and protein, Heinegard (11) has published very useful procedures which employ the Technicon AutoAnalyzer I. In this paper, a new method for the separation and quantitation of hexosamines, necessary modifications of some methods (i.e. those using the Technicon AutoAnalyzer II), and the adoption of a number of already published modifications to give a recommendable method (i.e. for the column chromatographic separation and quantitation of neutral sugars) are reported. All procedures described have been satisfactorily in use for at least 2 years in this laboratory. The use of some of these methods for the analysis of proteoglycan fractions has been reported recently (12). 539

0003-2697/78/0842-0539$02.00/O Copyright 8 1978 by Academic Ress, Inc. Ail rights of reproduction in any form reserved.

540

FORD

MATERIALS

AND

BAKER

AND METHODS

1. Amino Acid Analysis Apparatus. The apparatus included a Beckman Amino Acid Analyzer, Model 119, a column (53 x 0.9 cm) of Beckman PA 28 resin, vials (5 ml, Wheaton Gold Band), and a Millipore filter (0.22 CL) and plastic holder (5X 0601300). Reagents. Ninhydrin reagent was prepared as detailed in the Beckman Model 119 manual, July 1974, except for the use of titanous trichloride (7.5ml ampoule from Pierce Chemical Co.), which replaced stannous chloride (1.5 g). Buffer 1, sodium citrate buffer (0.2 N in Na+), pH 3.25, was prepared from the Beckman concentrate by appropriate dilution. Buffer 2, sodium citrate buffer (0.2 N in Na+), pH 4.30, was prepared from a Beckman concentrate, pH 4.25, by adjustment of pH with 5 N NaOH and appropriate dilution. Buffer 3, sodium citrate buffer (1.0 N in Na+), pH 6.40, was prepared according to directions in the Model 119 manual. “Diluting buffer”, pH 2.2, was also prepared according to directions in the Model 119 manual. Liquified phenol (Fisher Chemical Co.) was added to buffers 1,2, and 3 at 1.O ml/liter. Sodium hydroxide (0.2 M) contained disodium EDTA (Mallinckrodt, 0.5%). Amino acid mixture (0.5 mrvr in each amino acid) was prepared by diluting 2.0 ml of standard amino acids (Sigma AA S-18, 2.5 mM), 0.5 ml of norleucine (10 mM), and 0.5 ml of S-carboxymethylcysteine (10 mM) to 10 ml with diluting buffer. Hydrochloric acid was Aristar grade (Gallard Schlesinger). Procedure. For hydrolysis, a proteoglycan sample (1 .O mg dry weight), norleucine (0.3 ml of 0.5 mM), water (0.7 ml), and Aristar hydrochloric acid (1 .O ml) are sealed in a vial in vacua and heated at 105°C for 20 hr. Hydrolysates are filtered through a Millipore filter (porosity, 0.22 pm) prior to rotary evaporation to dryness. (The filtration of proteoglycan hydrolysates is especially important, as some humin particles are commonly present and must be removed to avoid subsequent clogging of the chromatographic column.) Samples in diluting buffer, pH 2.2 (0.25 ml), are applied to the column via a Beckman automatic sample injector (ASI 30). The conditions employed for the chromatographic separation are detailed in Fig. 1. 2. Hexosamine

Analysis

Apparatus. The apparatus included a Beckman amino acid analyzer, Model 120 C, and a column (55 x 0.9 cm) of Aminex A-4 resin (Bio-Rad). Reagents. Sodium citrate buffer (0.35 N in Na+), pH 5.28, was prepared and using the appropriate dilution from a concentrate (Pierce Chemical Co.) Pyrex tubes (16 x 150 mm). Diluting buffer, pH 2.2 was prepared as

ANALYSES

OF PROTEOGLYCANS

541

FIG. 1. A single column method for the analyses of amino acids and hexosamines in (A) a standard mixture (0.1 pmol of each residue) and (B) a proteoglycan (AID,) hydrolysate (1 .O mg). The analyzer is programmed (from the time of sample injection) as follows: 0 min, Buffer 1; 65 mitt, Buffer 2; 115 min, Buffer 3; 135 min, temperature to 65°C; 222 min, NaOH/EDTA and temperature to 54.5”C; 229 min (until 259 min), Buffer 1. The flow rate is 70 mYhr for buffers and 35 mhhr for ninhydrin.

above. Sodium hydroxide (0.2 M) contained EDTA (0.5%). Hexosamine standard was composed of a mixture of glucosamine hydrochloride (2.5 mM) and galactosamine hydrochloride (2.5 mM) at pH 2. Both sugars were purchased from Pfanstiehl, Waukegan. Hydrochloric acid was Aristar grade. Procedure. Proteoglycan samples (approximately 0.2 mg dry weight) for hydrolysis are weighed into 150 x 16-mm tubes, 2 ml of 6 M HCl are added, the tubes are capped with marbles, and are heated at 105°C in an oil bath for 7 hr. Any humin is removed by passage through a Millipore filter (0.22 pm), the hydrolysate is taken to dryness by rotary evaporation, the residue is dissolved in diluting buffer pH 2.2 (1.5 ml), and a 1 .O-ml sample is loaded on the column via an automatic sample injector (Chromatronix). The

FORD AND BAKER

542

conditions Fig. 2.

employed

for the chromatographic

separation

are detailed in

3. Neutral Sugar Analysis Apparatus. The sugar chromatography system and related equipment (except where otherwise stated) were obtained from Technicon Instruments Corp. The column provided was packed with Chromobeads (Type S) to a bed size of 70 x 0.6 cm. A constant-temperature water circulator (Haake, Type FJ) maintained the water jacket of the column at 54°C. Reagents. The buffers and gradient system employed for the fractionation of the sugar-borate complexes were as described by Hough et al. (S), but for completeness, full details are given here: 10% (w/v) aqueous potassium tetra borate was kept in a polypropylene bottle and fed to the column through an “in-line filter” (Evergreen). The borate buffers listed in Table 1 were titrated to pH 7.00 with 2 M sodium hydroxide. The gradient for elution of the column was generated from seven compartments of an Autograd. The makeup of this gradient, as recommended by Hough et al. (8), is reproduced in Table 1. A 0.1% (w/v) solution of orcinol (Fisher Chemical Co.) in cold 70% (v/v) aqueous sulfuric acid (Fisher Chemical

TIME

(MINUTES)

FIG. 2. The separation and quantitation of glucosamine and galactosamine in (A) a standard mixture (0.1 pmol of each amino sugar) and (B) a proteoglycan (AID,) hydrolysate (150 pg). The analyzer is programmed as follows (from the time of sample injection): 0 min, buffer (0.35 N Na+, pH 5.28); 80 min, ninhydrin pump and recorder on; 120 min, switch from buffer to NaOH/EDTA; 140 min, ninhydrin pump and recorder off, and regenerate with buffer (pH 5.28) for 40 min. Total time required: 180 min. The flow rate is 68 ml/hr for buffer and 34 ml/hr for ninhydrin. The temperature is 55°C.

ANALYSES

543

OF PROTEOGLYCANS

Co., ACS grade) was prepared according to the directions of Kesler (7). This reagent, 500 ml, was used for each run and was prepared fresh daily. The 70% sulfuric acid may be prepared in bulk and stored at 4°C. Aqueous trifluoroacetic acid, 2.0 M, was also used. Standard sugars included a mixture containing 400 &ml of rhamnose, mannose, fucose, galactose, xylose, and glucose (obtained from Pfanstiehl) which was prepared from stock solutions (4.0 mg/ml) of each sugar. All sugar solutions were dissolved in benzoic acid-saturated water and stored at - 15°C. The internal standard used was cellobiose (400 *g/ml). Procedure. In the analysis of glycosaminoglycans and proteoglycans, samples (approximately 2 mg) are hydrolyzed in 2 M trifluoroacetic acid at 105°C for 3 hr. After hydrolysis and cooling, 50 ~1 of cellobiose (20 pg) are added, the mixture is lyophilized, and the sample is taken up in water (0.2 ml) for application to the column. The column is prepared for chromatography by pumping (35 ml/hr) 10% tetraborate for 4 hr and 0.1 M H,BO,, pH 7.0, for 1 hr. Then, the sample (0.2 ml) is applied to the column and forced into the resin bed under nitrogen pressure. Sugars are separated by subsequent passage of the gradient given in Table 1 through the system illustrated in Fig. 3. The separation of a standard mixture of sugars is shown (Fig. 4). 4. Automated

Analysis

of Protein

Apparatus. The basic equipment used (i.e., the Technicon AutoAnalyzer II) is the same for this and the following two procedures, and will only be detailed here. The equipment includes the sampler IV, a proportioning pump III, a cartridge kit, type A, and a calorimeter, equipped with 500- to 900-nm spectral range phototubes for this method, but with the conventional phototubes in other methodologies. Other components of the apparatus include the AutoAnalyzer II recorder and the TABLE GRADIENT

Autograd chamber 1 2 3 4 5 6 7

SYSTEM

0.1 M H,BO,, pH 7.00 (ml)

I

FOR ELUTION

OF SUGAR COMPLEXES

0.2 M H3B03, pH 7.00 (ml>

50 35 25 25 25

0.2 M

H,B0,/0.2 M NaCI, pH 7.00 (ml)

15 25 25 25 50 50

FORD

AND

BAKER

FIG. 3. System for separation and analysis of neutral sugars. AU flow rates in this and subsequent flow diagrams are given in milliliters per minute.

modular digital printer. Other accessories (e.g., nipple connectors, tubing, etc.) were also obtained from Technicon Instrument Corp., Tarrytown, New York. Reagents. The reagents employed, as for the two following procedures, are as described by Heinegard (11) and were prepared fresh daily. Alkaline copper reagent is prepared as follows: 10% (w/v) aqueous sodium tartrate (1 part), 5% (w/v) aqueous cupric sulfate (1 part), 10% (w/v) aqueous sodium carbonate containing 2% (w/v) sodium hydroxide (100 parts). This reagent is consumed at the rate of 9.6 ml/hr. For the Folin & Ciocalteau

1

1

I

1

I

I

D

I

TIME (MINUTES)

FIG. 4. The separation of a mixture of standard sugars: rhamnose, mannose, fucose, galactose, xylose (40 pg of each), and cellobiose (20 pg).

ANALYSES

545

OF PROTEOGLYCANS

reagent (Fisher Chemical Co., SO-P-24), the stock reagent is diluted with an equal volume of water and 6 ml of this reagent are required per hour. Protein standards are prepared by diluting a stock aqueous solution of bovine serum albumin (10 mg/ml) with water to give 10, 20, 30,40, and 50 mg of protein/ml of standard solutions. Procedure. The analytical scheme illustrated in Fig. 5 is employed. No metal nipple connectors should be used in this system. The sampler is controlled by the digital printer for a sampling rate of 60 samples per hour and a wash time of 12 sec. As the calorimeter is fitted with 500 to 900 nm spectral range phototubes, a 750-nm filter, which is close to the absorbance maximum of the colored product may be used. Commonly, 1 ml of sample is pipetted into each sampler cup (0.64 ml is required for analysis). The wash solution is always identical to the sample solvent. The 30 kg/ml protein standard is used to set the recorder at 30% of maximum response. At the same time, the digital printer is adjusted to this protein concentration. Then, the protein content of subsequent samples which feed through the system is obtained as a printout in micrograms of protein per milliliter. The full range of standard protein solutions is run at the beginning and again at the end of a series of analyses. Although there is not strict linearity between concentration of protein and calorimeter response (as with other procedures based on the Lowry method), protein concentrations of up to 50 &ml can be determined accurately (Fig. 5, inset).

Ii3 2 10 turn mixing coils i

0.32;

1 Air

0.601

:

0.161

I Alkaline

0.10 I

I FolinCiocaltaau

SAMPLER

Copper

IV

50 pg/ml 40 pglml

x,rA..--LII~, j Hot4JlW RECORDER 1 in/min

II MO DULAR DffilTAL PRINTER

~~~~ COLORIMETER FILTER 750 nm FLOWCELL 15 nm PHOTOTUBE 5009OOnm

30 @ml 20 pglml r,..., ” 4 I 1

I 2

8 3

I 4

I 5

6

TIME (minutes)

FIG.

obtained

5. AutoAnalyzer II system for determination with a series of bovine serum albumin

of protein. standards.

Inset is the recorder

response

546

FORD AND BAKER

5. Hexose Analysis Reagents. Anthrone, 0.2% (w/v) in 91% (v/v) aqueous sulfuric acid, is used at a rate of 110 ml/hr. Standard hexose solutions containing 10,20,30, 40 and 50 pg of galactose/ml in water were prepared from a stock galactose solution (1 .OO mM in benzoic acid-saturated water and stored at - 15°C). Procedure. The analytical scheme illustrated in Fig. 6 was employed. Under the conditions shown, 0.24 ml of sample is required for each determination, so a 0.5-ml aliquot pipetted into the sampler cup is a comfortable excess. The recorder response is still linear with concentrations up to 80 pg galactose/ml (Fig. 6, inset). Uranic acid gives some interference (glucuronolactone gives 16% of the color yield of an equimolar amount of galactose). 6. Uranic Acid Analysis Reagents. The reagents used for uranic acid analysis are 0.05% (w/v) carbazole in tetraborate/sulfuric acid and uranic acid standards. Sodium tetraborate (0.025 M) in concentrated sulfuric acid was prepared in bulk and stored at 0°C. Carbazole (recrystallized twice from hot benzene) was dissolved in the tetraborate/sulfuric acid to give a 0.05% (w/v) solution, prepared fresh daily, and kept on ice during use. Approximately 130 ml of reagent are required per hour. Uranic acid standards containing 7, 14, 21, 28, and 35 ,ug of glucoronolactone/ml in water were prepared from a stock solution (1.00 mM glucuronolactone in benzoic acid-saturated water) and stored at -15°C.

1.25 ,AFi

‘ml

60 50 CARTRIDGE KfTA

40 30 COLORIYETER’ FILTER 570 nm FLOWCELL 15 m m PHOTOTUBE 380660 M

20 10 0 1

2345678 TIME (minutes)

FIG. 6. AutoAnalyzer II system for determination of hexose. Inset is the recorder response obtained with a series of galactose standards.

ANALYSES

OF

547

PROTEOGLYCANS

Procedure. The analytical scheme is illustrated in Fig. 7. For each determination 0.19 ml of sample is required. Linearity between glucuronolactone concentration and recorder response is still obtained at the highest standard concentration (i.e., 35 pg/ml, Fig. 7, inset). There is some interference by hexoses (galactose gives 11% of the color yield of an equimolar amount of glucuronolactone). RESULTS

AND DISCUSSION

1. Amino Acid Analysis

Methods for the amino acid analysis of proteins abound. Those methods which have been designed for the analysis of connective tissue proteins have mainly been concerned with the separation of amino acids, including the unusual ones, from collagen and elastin (13,14). A few (l-3) have been employed in the analysis of proteoglycans. The Beckman single column hydrolysate method was modified in order to separate the hexosamines, which occur in large amounts in proteoglycans, as widely as possible from neighboring amino acids without causing unacceptable broadening of any amino acid peak. Employing the system detailed in Fig. 1, an amino acid mixture is separated as illustrated (Fig IA). Although both hexosamines are eluted from the column by Buffer 3, they are separated one from the other. All amino acids present are adequately separated, and a complete cycle requires 260 min. A hydrolysate of chondroitin sulfate proteoglycan (AID,), similarly separated (Fig. lB), shows comfortable separation of glucosamine from phenylalanine and of the large amount of galactosamine from histidine.

waste

4

1.46 IAF: :L-.1 :

I

I t 40

CARTRIDGE KIT A

35 pglml

-

PHOTOTUBE

FIG. 7. AutoAnalyzer response obtained with

360~ 660 nm

II system for determination a series of glucuronolactone

2

of uranic standards.

3 4 TIME (minutes)

acid.

Inset

5

6

is the recorder

548

FORD

2. Hexosamine

AND

BAKER

Analysis

The results for glucosamine and galactosamine from an amino acid run are used by some workers to estimate a sample’s glucosamine to galactosamine ratio, although considerable destruction of hexosamines occurs under the normal conditions of protein hydrolysis. Milder conditions of hydrolysis are necessary for a closer approach to absolute values of hexosamine content. As a separate analysis is required, it is convenient to use a simpler, accelerated column system, in which separation of the two hexosamines from other amino acids and each other is increased, but where no effort need be made to maintain separation of individual amino acids. In the system presented here, the separation and quantitation of glucosamine and galactosamine have been based upon a report (15) that they are well separated on a column of Beckman PA-35 resin in a sodium citrate buffer (0.35 N in Na+ at pH 5.28). We have chosen to use a relatively cheap resin (Aminex A-4) eluted with this one buffer. Under the conditions described, a wide separation has been achieved (glucosamine and galactosamine emerge at 114 and 128 min, respectively, Fig. 2A), which even allows for the accurate estimation of one hexosamine in the presence of a loo-fold excess of the other. To permit the continuous use of this system for analyzing multiple samples, the analyzer was controlled using a BioCal modification kit. The important features of this control are given in Fig. 2. The procedure lends itself to the routine, automated analysis of proteoglycan hydrolysates at the rate of 8 per day. A similar system employing high pressure liquid chromatography has recently been described (16), which likewise permits analysis of samples rich in hexosamines. The time course of galactosamine release from chondroitin sulfate has been studied. At 105°C in 4 N HCl, the optimal yield of galactosamine occurred after 10 hr of hydrolysis, whereas in 6 N HCl, the yield of galactosamine was 8% higher, and optimal release occurred at 7 hr. If hydrolysis time is less than 7 hr. a shoulder is seen on the glucosamine peak, but at times in excess of 7 hr, yields of both hexosamines suffer. The separation of a typical chondroitin sulfate proteoglycan (A,D,) hydrolysate (7 hr in 6 N HCl at 105’C) is given in Fig. 2B. 3. Neutral

Sugar Analysis

Only minor modifications have been applied to the autoanalyzer methodology recommended by Technicon. The schematic diagram (Fig. 3) illustrates the system employed. For pulse suppression, we have found that a constriction (0.03-mm-i.d. acidflex tubing) in the line between sensitizing lamp and calorimeter is effective. Also, to increase sensitivity, a greater proportion of the column effluent was taken through the heating bath to the

ANALYSES

549

OF PROTEOGLYCANS

calorimeter and proportionally less allowed to go to waste (Fig. 3). Passage of the gradient (Table 1) resulted in the separation of sugars illustrated in Fig. 4. All sugars present in the mixture are satisfactorily resolved, including those neutral sugars which are known to be present in connective tissue glycosaminoglycans. Glycoprotein or glycosaminoglycan samples commonly have been hydrolyzed in dilute mineral acid and desalted with ion exchange resins prior to analysis in this manner (e.g. Ref. 8). We have found that aqueous trifluoroacetic acid hydrolysates of samples, after lyophilization, may simply be applied directly to the column without adversely affecting the separation of the common sugars. 4,5,6. Automated

Analyses

of Protein,

Hexose,

and Uranic Acid

Necessary changes to the AutoAnalyzer I methods of Heinegird (11) have been made to permit their use on the Technicon AutoAnalyzer II. In addition we have incorporated the advantages of a single sampling and wash rate in all three procedures. The AutoAnalyzer II system has the capacity to analyze simultaneously samples for protein, hexose, and uranic acid. The choice of anthrone, rather than phenol or orcinol, which have all been employed in automated methods for the determination of hexoses (I 1,17,18), is dependent upon a number of factors. The orcinol method suffers from lack of specificity and interference by chloride ions. Phenol has the advantages of cheapness and availability in a pure form but gives lower color yields with hexoses than anthrone and a high color yield with uranic acid. Galactose and mannose give approximately the same, but glucose a relatively greater, color yield in the anthrone reaction (19). With many glycoproteins and proteoglycans which contain no glucose, this poses no problems. Otherwise, the unrecognized presence of glucose in samples will lead to anomalously high estimates of hexose content by the anthrone method. 7. Analysis

of Sulfate

Attempts have been made to automate the method of Terho and Hartiala (20) for the analysis of ester sulfate. Problems with reagent precipitation in the transmission tubing have been encountered and could not be solved by altering the type of tubing, concentration of reagents, inclusion of detergents, or manner of mixing reagents. Therefore, in this laboratory the unmodified manual method of sulfate estimation is employed (20). ACKNOWLEDGMENTS This

work

was supported

by NIH

Grants

DE 02670 and HL

11310.

550

FORD AND BAKER

REFERENCES 1. Mashbum, T. A., Jr., and Hoffman, P. (1970)Anal. Biochem. 36, 213-221. 2. Heineglrd, D. (1972) Biochim. Biophys. Acfa 285, 181-192. 3. Hascall, V. C., Riolo, R. L., Hayward, J., Jr., and Reynolds, C. C. (1972)5. Viol. Chem. 247, 4521-4528. 4. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. 5. Khym, J. X., and Zill, L. P. (1952) J. Amer. Chem. Sot. 74, 2090-2094. 6. Technicon Chromatogr. Corp. (1966) Technicon Sugar Chromatography Brochure, Arsley, New York. 7. Kesler, R. B. (1967) Anal. C’hem. 39, 1416-1422. 8. Hough, L., Jones, J. V. S., and Wusteman, P. (1972) Carbohyd. Res. 21, 9-17. 9. Spiro, R. G. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, Part B, pp. 3-43, Academic Press, New York. 10. Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, Part B, pp. 63-73, Academic Press, New York. 11. Heineglrd, D. (1973) Chem. Ser. 4, 199-201. 12. Baker, J. R., and Caterson, B. (1977) Biochem. Biophys. Res. Commun. 77, l-10. 13. Miller, E. J., and Piez, K. A. (1966) Anal. Biochem. 16, 320-326. 14. Starcher, B. C., Wenger, L. Y., and Johnson, L. D. (1971)J. Chromatogr. 54,425-427. 15. Liu, T. Y., and Chang, Y. H. (1971)J. Biol. Chem. 246, 2842-2848. 16. Georgiadis, A. G., Coffey, J. W., Hamilton, J. G., and Miller, 0. N. (1975) Anal. Biochem. 67, 453-461. 17. Brummel, M. C., Mayer, H. E., Jr., and Montgomery, R. (1970) Anal. Biochem. 33, 16-27. 18. Judd, J., Clause, W., Ford, J., van Eys, J., and Cunningham, L. W. (1%2) Anal. Biochem. 4, 512-514. 19. Spiro, R. G. (1966)in Methods in Enzymology (Colowick, S. F., and Kaplan, N. O., eds.), Vol. 8, pp. 3-26, Academic Press, New York. 20. Terho, T. T., and Hartiala, K. (1971) Anal. Biochem. 41, 471-476.