Characterization of glycosaminoglycan-alcian blue complexes by elution from cellulose acetate utilizing different MgCl2 concentrations

ANA1

YTICAL

BIOCHEMISTRY

107. 393-405 (1980)

Characterization Complexes Utilizing LUCJAN

of Glycosaminoglycan-Alcian Blue by Elution from Cellulose Acetate Different MgCI, Concentrations

HRONOWSKI’

TASSOSP.

.~ND

Received

January

ANASTASSIADES’

25. 1980

Glycosaminoglycans subjected to electrophoresis on cellulose acetate are stained by Alcian blue in the presence of different MgCI, concentrations. The procedure described solubilizes and elutes off the cellulose acetate strip glycosaminoglycans with critical electrolyte concentrations lower than the concentration of MgCI,. leaving stained on the strip glycosaminoglycans with higher critical electrolyte concentrations. This technique is particularly useful for sulfated glycosaminoglycans that are incompletely separated by electrophoresis and is applicable to the partial characterization of glycosaminoglycans down to a 0. I-wg range allowing practical quantitative studies of radioactively labeled glycosaminoglycans produced by low-density cell cultures. The staining technique is examined with respect to the specificity of the glycosaminoglycans eluted, and the nature of the interactions in the staining process is discussed. In addition. a simplified procedure is described for the characterization of glycosaminoglycans using testicular hyaluronidase (EC 3.3.1.35) and chondroitinase ABC (EC 4.7.2.4). which can be used in conjunction with the salt elution technique.

In a previous communication ( I) we had reported an improved method for the quantitation of small amounts of glycosaminoglycans separated by electrophoresis on cellulose acetate, stained with Alcian blue, and dissolved in a dimethylsulfoxide solution. Present electrophoretic techniques (2-7) allow adequate separation of hyaluronic acid from the sulfated glycosaminoglycans, but clear-cut separations between a number of the sulfated glycosaminoglycans are often not feasible or incomplete under optimal one-dimensional electrophoretic conditions. This problem is due not only to the polydispersity of chain length, but also to the presence of copolymeric structures (8). Deg’ Present address: Department of Chemistry, University, Kingston, Ontario. Canada. ’ Request for reprints should be addressed Anastassiades Director, Rheumatic Diseaxe partment of Medicine. Queen’s University. St.. Kingston. Ontario. K7L 357. Canada.

Queen‘s to Dr. T. Unit. De26 Barrie

393

radative enzymatic methods (7,9) for glycosaminoglycans constitute a useful approach to this problem. However, the application of the critical electrolyte concentration technique (10.11) to one-dimensional electrophoresis for glycosaminoglycans offers an additional simple and informative approach to the characterization of these macromolecules. We report a simple procedure where glycosaminoglycans separated by electrophoresis on cellulose acetate are stained by Alcian blue in the presence of different concentrations of MgCl,. The procedure elutes GAGS” whose critical electrolyte ,’ Abbreviations used: GAG, glycosaminoglycan: DMSO, dimethylsulfoxide; Hya. hyaluronate: Ch6-S. chondroitin 6-sulfate: Ch4-S, chondroitin 4-sulfate; Des. dermatan sulfate; Hep. heparin; HeS. heparan sulfate; KeS- 1, keratan sulfate- 1: KeS-2. keratan sulfate-?; ddd. deionized double-distilled water; PAG. potential anionic groups; PPO. 2.5.diphenyloxazole: POPOP, I’-bis[?-(5.phenylorazolyl)]benzene.

0003.X597/80/140393-13$03.00/O CopyrIght All right\

,e 19X0 by Acadrmic Press. Inc hit’ reproductxtn in an) form rrvsrved.

394

HRONOWSKI

AND

concentration is below that of the MgCI, solution and facilitates the quantitative characterization of small amounts of GAGS down to the O.l-pg range. It is particularly useful in the identification and partial characterization of certain of the sulfated glycosaminoglycans that comigrate or migrate very close to each other during electrophoresis on cellulose acetate strips (e.g., dermatan and heparan sulfate). By systematically varying the salt concentration during the dye-glycosaminoglycan interactions and after the dye-glycosaminoglycan complex has formed on the strip, information can be obtained about the nature of the elution process and the heterogeneity of some of the glycosaminoglycans. In addition, the relationship of charge density and unit weight of glycosaminoglycans to dye uptake can be examined. A simplified enzymatic procedure applicable to this system is also described that may be used as an adjunct to identification of the glycosaminoglycans. The method is suitable for the study of GAGS produced by connective tissue cells growing in small tissue culture dishes or in sparse cultures and is useful for comparing the physical characteristics of radioactively labeled glycosaminoglycans synthesized in culture to exogenously added standards.

ANASTASSIADES

cress, ribonuclease A (EC 2.7.7.16) from bovine pancreas. hyaluronidase (EC 3.2. I .3S) type IV from bovine testes, chondroitinase ABC (EC 4.2.2.4). papain (EC 3.4.22.2) P-3 125, ribonucleic acid type VI from Towlrt yeast, deoxyribonucleic acid type I from calf thymus, chondroitin sulfate type C from shark cartilage, and chondroitin sulfate type A from whale cartilage were purchased from Sigma Chemical Company. Albumin bovine fraction V and glycine were purchased from Nutritional Biochemicals Corporation. Acid phosphatase was from Worthington Biochemical Corporation. Pronase. phytohemaglutinin M (PHA) from Pl~rr.sc~olrr.~ \~trlgrrri.s~, and concanavalin A were purchased from Calbiochem. 25Diphenyloxazole (PPO) and IT-bisl2-(5phenyloxazolyl)]benzene (POPOP) were purchased from New England Nuclear. t--Ascorbic acid. L-proline, Dulbecco’s modified Eagle’s medium, fetal bovine serum (heat inactivated), penicillin 10,000 u/ml, streptomycin 10,000 pglml, and trypsin 0.25% were purchased from Grand Island Biological Company. Ethanol was 99%. All other chemicals were of the highest purity available. G!\‘c~c~.snt77it7o,~l~c~rinrc
GLYCOSAMINOGLYCAN TABLE ANAI.YTICAL

A&D

PHYSICAL

acid”,’ acid”.’

Hexosamine”.” Sulfate’ .’ Galactose”,” Molecular ” All

of the

HT

HeS

47.2

34.8

32.0

43.8 38.3

35.7 31.4

33.1 17.9

IS.5 33.8

40.5 34.9

49.2 42.8

3l.S

23.3

28.’

standards, with

from

Institutes by weight.

” By carbazole ” By a modified Ch4-S

0.90 0.9

0.98 1.4

29,000

in Table

glycan reference data are reproduced the National ” Percentage

SAL IS OF GLYCOSAMI~.OGI.~CANS” DeS

tr. 230.000 data

FOR SOI)IUM Ch4-S

0.00 weight’

DATA

I

Chh-S

Hya Hexuronic Hexuronic

395

CHARACTERIZATION

I except

which permission.

1.13 0.3

IS ,000 for

the

2.71 tr.

45.000

second

hexuronic

were kindly provided by These reference standards

I

Kes-2

1.9

0.97 0.6

14.000 acid

Dr\.

Kes-

-

29.3

78.0

1.17 36.9”

I.76 ‘0.6

16.000

value

were

supplied

with

glycosamino-

M. B. Mathews and J. A. Cifonelli and were prepared under Contraci NO-AM-52205

the

of Health.

method of Dische Elson-Morgan

t71). method

(23)

with

correction

for

losj

on

hydrolysis

for

Hya.

Ch6-S.

and

only. ’ Molar ratio ’ By method

of ester sulfate to hexosamine. of Dodgson et ul. (24) as modified

” By chromatographic procedure ’ By method of Dische (28). Viscosity average molecular 171 and

molecular

’ Determined

of Cifonelli weight.

,U,

by

Muir

(25)

and

Cifonelli

~‘1 trl. (16).

(11 trl. (27). (79-3

I). estimated

from

relationships

hetween

intrinsic

viscosity

weight. by

the

present

author

using

the

Bitter

and

Muir

method

( 12).

of 7km’ surface area. The cells were centrifuged as described above. The acetone grown in Dulbecco’s modified Eagle’s me- wash was discarded and the precipitate was dium containing 10% fetal calf serum and air-dried overnight. Commercial papain (Psupplemented with 3.7 g NaHCO:,, 0.05 g 3125 Sigma) suspension was dissolved at ascorbic acid. 0.40 g proline, 0.80 g glycine. 1.5% (v/v) in a buffer consisting of 0.075 M and 10 ml of penicillin-streptomycin solu- EDTA and 0.005 M cysteine. pH 7.2. Four tion per 1 liter of medium. After the cells milliliters of this solution was added to the reached confluency they were incubated for dried precipitate and the digestion was al48 h with 2.5 &i of D-[1-“C]g]ucosamine. lowed to proceed for 24 h at 6YC. The (sp act, 45-60 mCi/mmol. New England papain digest was diluted with 10 ml of Nuclear Co.). The cells were then grown double-distilled, deionized (ddd) water and for an additional 48 h with 2.5 &i of D0.4 ml of 55? cetylpyridinium chloride in ] I-‘-‘C]glucosamine in fresh medium. This 0.2 M Na,SO, was added. After I h at latter medium was then used for extraction 37°C. the flocculent precipitate was colof the labeled glycosaminoglycans. lected by centrifugation at IS,OOO~r:for 10 The glycosaminoglycans were isolated by min at room temperature. The glycosaminothe following modification of previously de- glycans were converted to their sodium scribed procedures ( 13.14). Four volumes of salts by dissolving the precipitate in 2 ml ethanol was added to the medium in 30-m] of 2 M sodium acetate-ethanol (100: 15. v/v) Corex tubes and after 1 h at room tempera- solution. pH 7.0. After incubation for 30 ture the precipitate was collected by cen- min at 37°C. 6 vol of ethanol was added trifugation at lO,OOO
396

HRONOWSKI

AND

collected by centrifugation at 15.000~ for IO min at 4”C, air-dried. and transferred to 3-ml tubes by washing the 30-ml Corex tubes three times with 0.2 ml ofddd water. Following lyophilization, the glycosaminoglycans were redissolved in an appropriate small volume (usually Z-200 ~1) of ddd water and measured aliquots were subjected to analysis.

ANASTASSIADES

Mark I I1 liquid scintillation counter (Searlr Analytic Inc.). The cellulose acetate strip containing the separated and stained glycosaminoglycana was cut into 2- to IO-mm-wide strips. The strips were suspended in IO ml of scintillation solution consisting of 4 g PPO and 0.3 g POPOP per liter of scintillationgrade toluene (13). After the counting ol the radioactivity the same strips and their corresponding blanks were dissolved in a dimethylsulfoxide solution containing 0.5c/; (v/v) H,SO, in order to quantitate the Alcian blue-glycosaminoglycan complexes. as described previously (1). Digo.vlic~ti tc.stic.rrltrt.

I

2

.3

4

5

MgCI, FIG. reference

I.

Elution standards.

cosaminoglycan

6

profile? of Comtant was

7

Cancn

applied

8

9

1011

12

(M 1

the glycosaminoglycan amount of each on

cellulose

gly-

acetate.

electrophoresed for tion containing 0.X (v/v), O.lC; CH,,COOH centration. The rinse

5-10 min and 5taincd in a soluAlcian blue (wiv). IO? EtOH (v/v). and variable M&I, consolutions were identical with the

staining

hut

Amounts uronate. droitin heparan sulfate-l.

solutions.

without

the

Alcian

blue.

of each glycosaminoglycan used were: hyal2.81 pug: chondroitin I-sulfate. 3.66 pug: chonh-sulfate. 3.32 pg: dermatan sulfate. 3.00 pg; sulfate. 3.61 ~g: heparin. 4.51 pg: keratan 3.61 pp: and keratan sulfate-?. 3.78 /LL~.

All strip\ containing for detail\).

were dissolved 0.094 hf H,SO,

in 2 ml of dimrthylsulfoxide (see Material< and Methods

,~!\‘c.o.\.rrtttitto,e!\‘c’trtt.\ (EC

3.2.

il,ilil I.351

titrcl

ABC’ (EC‘ 4.22.4).

Five hundred units of commercial hyaluronidase (X80 NF unitsimg) was dissolved in 1 ml of buffered solution consisting of 0. I5 M NaCI in 0.1 M sodium acetate, pH 5.0 (IS). Fifty microliters of this enzyme solution was added to 40-60 pg of glycosaminoglycan standards and incubation was allowed to proceed at 37°C for 8 h. followed by :I Imin incubation in a boiling water bath. For chondroitinase ABC, Tris-HCI buffer. pH 8.0. as had been described before (9) was prepared and 25 PI of this buffer containing 0.X p/ml of the enzyme was added to the glycosaminoglycans, also dissolved in 15 PI of the buffer. The reaction was carried out at 37°C for IO min and stopped by immersing in a boiling water bath for I-min. The enzymatic digests were lyophilized. and the samples dissolved In a known VOIume of ddd water. applied on cellulose acetate. electrophoresed, stained. and quantitated in a DMSO solution containing 0.09 M H,SO, ( I ). It should be noted that the above incubation conditions are applicable to purified standards, but less purified biological samples may require longer incubation times or increased enzyme concentrations. c.ltotttlt.oititttr.\c’

0

c$llir

/r~~trltr~~tttiLltr.s~,

Alcirrtl hl~rc .slcritliyq of tiotr,yi~c~o.strttiirlo,gl.w~tt tttrrc~t~ot~lol[~c~i~llrs. Nonglycosaminoglycan

macromolecules

were

dissolved

in a

GLYCOSAMINOGLYCAN

phosphate-buffered saline solution ( 16). concentration of these macromolecules 3-5 mg/ml unless otherwise indicated 5 ~1 samples were used for analysis procedures identical to those used for glycosaminoglycans.

The was and by the

RESULTS

E,‘lrrtic,rrPlwjilcA c~f‘riw Gl~c~o.srir~li~lo,~~l~c~rrll R&w~c~

Stcmtltrdv

Dye uptake by glycosaminoglycans separated on cellulose acetate is highly dependent on the salt concentration in the staining solutions. As seen in Fig. 1, dye uptake by the glycosaminoglycans increases initially as the MgCI, concentration of the staining solution is increased, reaching a maximum at 0.030 M MgCI, for hyaluronate and 0.200 M MgCI, for the sulfated glycosaminoglycans. The increase in dye uptake (from zero salt to the salt concentration giving maximal absorption) is only 509 for hyaluronate. but approximately twofold for the sulfated glycosaminoglycans, except for keratan sulfate-l and keratan sulfate-2, where dye uptake is increased threefold. This effect of salt concentration on dye uptake is not dependent on staining times. which were varied between 15 and 60

M&I, GAG Hya Ch4-S Chh-S DeS HeS HelJ KeS-I KeS-2

0 0.449 0.443 0.550 0.445 0.461 0.513 0.287 0.305

I\lr~fe. Constant Methods for further

amount details.

397

CHARACTERIZATION

min in three experiments. which yielded patterns identical to those in Fig. 1. As the MgCl, concentration is further increased the stained glycosaminoglycans start to elute off the cellulose acetate. Hyaluronate. whose staining is completely prevented at 0.1 hl MgCI, is eluted first. As the M&I, concentration is increased further chondroitin 4-sulfate and heparan sulfate are eluted. Staining of these two glycosaminoglycans is completely prevented at approximately 0.475 M MgCI?. At this concentration of MgCI,. staining by dermatan sulfate is still maximal. thus allowing a clean separation of heparan sulfate from dermatan sulfate. two glycosaminoglycans which often migrate close together on cellulose acetate electrophoresis. It will be noted that the cornea1 and skeletal keratan sulfate elution patterns are different and that heparin and dermatan sulfate have wide plateaus. Additional information about the dyeglycosaminoglycan interaction can be obtained if the glycosaminoglycans are stained in the presence of MgCIZ concentrations up to the concentrations giving maximal dye uptake and then rinsed in a rinse solution containing no MgCI, (Table 2). Under these circumstances. the final absorbance obtained for some of the glycosaminoglycans

concentration

moles/liter

0.0x

0.040

0.060

0.0x0

0. I00

0.300

0.500

0.370 a. 467 0.561 0.461 0.478 0.486 0.327 0.329

0.363 0.471 0.559 0.463 0.4YO 0.502 U.313 0.332

0.3 I I 0.47 I 0.564 0.456 0.499 0.460 0.327 0.34 I

0.163 0.463 0.543 0.484 0.505 0.473 0.340 0.344

0. I43 0.481 0.561 0.480 U.JYZ 0.433 0.343 0.343

0.161 0.326 0.515 0.447 0.434 0.396 0.311 0.328

0. 1 Xl 0.053 0.243 0.449 0.088 0.337 0.333 0.177

of each glyco~aminoglycan

WI!, applied

on cellulose

acetate.

See Material<

and

398

HRONOWSKI

AND

is the same as if they were stained and rinsed in the absence of salt. This behavior applies to chondroitin 4-sulfate. chondroitin 6-sulfate, heparan sulfate, dermatan sulfate, keratan sulfate- 1. and keratan sulfate-2. When these glycosaminoglycans were stained in the presence of MgCl? concentrations that were higher than those for maximal dye uptake, lower absorbances were obtained. However, hyaluronate and heparin demonstrate a different behavior (Table 2). Hyaluronate that is stained in the presence of MgCI, concentrations of up to 0.500 M still binds considerable dye if the strip is rinsed in the absence of salt. Heparin also follows a different pattern, in that absorbance is less than expected even before the maximal dye uptake plateau is passed.

Figure 2 compares the elution patterns of chondroitin sulfates from different sources. Chondroitin 4-sulfate from the notochord of

0

123456769

MgCI, FIG. different Amounts pressed equivalent method. notochord

2. Elution sources. of each in terms (0)

profiles

Corm

(Ml

of chondroitin

(see legend to Fig. chondroitin sulfate of micrograms of

determined by the Bitter Chondroitin 4-sulfate. of the river sturgeon.

sulfates

ANASTASSIADES

the river sturgeon elutes off in the presence of lower salt concentration than the chondroitin 4-sulfate from whale, whose elution pattern resembles that of the chondroitin 6-sulfate from the cranial cartilage of the river sturgeon. The chondroitin 6sulfate from shark cartilage elutes off at a higher MgCI, concentration than the chondroitin 6-sulfate from the river sturgeon. Ellrtion

Prc!file

of‘ ‘T-Lrrhrlrtl

As was the case for the glycosaminoglycan reference standards (Fig. 1) and for the commercial chondroitin sulfates (Fig. 2). dye uptake by the glycosaminoglycans synthesized by the tissue culture cells also increases with increasing MgCl? concentration up to a maximal value at 0.030 M MgCI, for hyaluronate and approximately 0.200 M MgCl, for chondroitin sulfate (Fig. 3). The radioactivity incorporated into the glycosaminoglycans, however, is maximal at zero MgCl, concentration and decreases only after the dye uptake peak is reached. The decrease in radioactivity then closely parallels the decrease in absorbance as the glycosaminoglycans are eluted off the cellulose acetate strip. The hyaluronate elution profile in Fig. 3 resembles closely the hyaluronate reference standard elution profife in Fig. 1 and the chondroitin sulfate elution profile lies in between that of chondroitin 6-sulfate from the river sturgeon and that of chondroitin 6-sulfate from shark cartilage (Fig. 2).

from

I for methods). used are exhexuronic acid and 1.21

Muir pg.

(I?) from

.Tc,rr~hirll?Nc,/rt.\

p/~tor~~c.hrrs: (a) chondroitin h-sulfate, I. 19 pg. from cranial cartilage of the river sturgeon, S. ~~lr~ro~nc.hrrs: (0) chondroitin sulfate type A. 1.36 Kg. From whale cartilage: (m) chondroitin sulfate type C. 1.61 pg. from shark cartilage.

Table 3 shows the amount of dye bound by the glycosaminoglycans at 0.030 and 0.200 M MgCI, concentrations. This is expressed both in terms of the glycosaminoglycan weight and in terms of its hexosamine content. The amount of Alcian blue bound

GLYCOSAMINOGLYCAN

was calculated using a standard curve for Alcian blue (I). The number of anionic groups was expressed per unit weight of glycosaminoglycan (Table 3) rather than per disaccharide unit, since at least some of the glycosaminoglycans, including heparin and heparan sulfate, have an apparent excess of hexuronic acid ( 17). In order to gain insight as to how dye binding correlates with the weight of the glycosaminoglycan, the hexosamine content and the charge density (defined as moles of potential anionic groups per kilogram of glycosaminoglycan), correlation coefficients (18) have been calculated from the data in Table 3. For 0.030 M MgCI, the correlation coefficient between the amount of dye bound per unit weight and the charge density (PAG) per unit weight is 0.718 and is significant at the 5% level. However, the correlation coefficients between the dye bound and the weight of the glycosaminoglycans or the hexosamine content are only 0.609 (not significant at 10%) and -0.084. respectively. For the 0.200 M MgCI, concentration rather different correlations are observed

-40 -36 -32

-28 -24

t

D~NSIT\

ON IHE

Moles of PAGi 1 kg of GAG

GAG Hya Ch4-S Ch6-S DeS HeS Hep KeSKrS-2

I

2.49 3.58 3.78 4.13 4.13 5.99 ?.I? 2.19

0.030

of dye boundil &I MgCI, 1.97 ’ 14 -.2.50 2.09 ’ ‘4 -.’ ?h -.I .47 I .4l

-4 b-

cs

HYO

3

GL\~COSAMINOC;L’~(.,WS

Moles

I I ;

FIG. 3. Elution profile of glycosaminoglycans synthesized by rat muscle fibroblasts in the presence of n-[ I-“C]glucosamine. Samples (5 1.~11were applied at the origin (rl and electrophoresed for I h. Cellulose acetate strips for absorbance and radioactivity measurement were cut as shown. Same strips were used for both absorbance and radioactivity measurement (see legend to Fig. I and Materials and Methods). Absorbance: (0) Band III comigrates with hyaluronate (Hyal: (ml Band V comigrates with the chondroitin sulfate (CSl. Radioactivity: (83) Band III (Hya): (3) Band V (CSl; (i I Band IV.

TABLE CHARGE

399

CHARACTERIZATION

AI\”

Moots

OF AL.CI~N

M MgCI, -

2.76 2.73 2.Y8 ‘7.61 2.73 7.47 2.37

BOUND

Moles of dye bound/ mole of hexosamine

kg of GAG 0.200

BLUL

0.030

M MgCI,

0.200

M MgCI,

0.879

I .30 I.28 I .07 1.78 I.56 0.808 0.813

I .6O 1.40 1.53 I.49 I.89 1.36 1.36

.Yore. Moles of potential anionic groups (PAG) (carboxyl and sulfate) per I kg of glycosaminoglycan was calculated from data in Table I. In these calculations hexuronic acid given by the Bitter and Muir (12) method was used. Hexosamine content was provided with glycosaminoglycan standards (Table 1). (See Legend to Fig. 1 and Materials and Methods for staining and absorbance measurement procedure.) Moles of dye bound was estimated from an Alcian blue standard curve ( Il.

400

HRONOWSKI

AND

for the sulfated glycosaminoglycans. At this higher salt concentration the correlation coefficient between dye bound per unit weight and the PAG per unit weight is 0.63 1 (not significant at the 10% level), and for hexosamine content it is only 0.222, indicating that there is no correlation between dye binding and these two variables. However, at 0.200 M MgCl, the correlation coefficient between the amount of dye bound and the weight of the glycosaminoglycan is 0.840 (significant at the 2% level). Table 3 shows that the number of moles of dye bound per unit weight of sulfated glycosaminoglycan is relatively constant at 0.200 M MgCl, with a mean and SD of 2.66 +- 0.076 mol of Alcian blue bound/kg of glycosaminoglycan. The data in Table 3 thus suggest that at 0.200 MgCl,, dye binding by the sulfated glycosaminoglycans depends on the weight and not the type of glycosaminoglycan being stained. Effect of Alcian Blue Concentmtion and Staining Time on Dye Uptake by the GlycosaminoglycarIs Dye uptake by chondroitin 6-sulfate and hyaluronate is not dependent on dye concentration in the O.l-0.5% (w/v) range, as seen in Fig. 4. At lower dye concentra-

Alcian

Blue Concn

%( w/v)

4. Effect of Alcian blue concentration on the intensity of staining. Constant amount of each glycosaminoglycan was stained in solutions containing 10% ethanol (v/v). 0.030 M MgCl,. 0.1% acetic acid (v/v) and variable amounts of Alcian blue. Absorbances were measured in DMSO solution D ( 1). Hyaluronate (0) stained for 15 min. (x) for 30 min; chondroitin 6.sulfate (0) stained for 15 min, (+) for 30 min. FIG.

ANASTASSIADES

8 s $2 E a

3

.I 0

50

100

150

CH3COOH

200 Concn

800 (mM

em

900

)

FIG. 5. Effect of acetic acid concentration in the staining and rinsing solutions on the intensity of staining. Staining solution consisted of 0.2%~ Alcian blue (w/v), 10% ethanol (v/v), 0.030 M MgCI, and variable amounts of acetic acid. The rinsing solution is identical to the staining solution, but without the Alcian blue. Absorbance was measured 30 min after adding the stained bands to DMSO solution D (I). (0) Hyaluronate. (A) heparin, (0) chondroitin h-sulfate. (a) keratan sulfate- I.

tions the intensity of staining tends to be quite variable. The differences in dye uptake at 15 and 30 min are only 3.4% for hyaluronate and I .5$% for chondroitin sulfate, but these small increases in dye uptake are statistically significant at the 1 and 2% level. respectively. It should be noted that although dye uptake by glycosaminoglycans does not increase with higher dye concentrations in the range of 0.1-0.5s (w/v) the amount of dye remaining as background on the cellulose acetate strip increases one- to twofold at the higher dye concentrations, thus decreasing the sensitivity of the method.

The effect of acetic acid on dye uptake or final staining intensity by the different glycosaminoglycans is highly dependent on the type of charge groups present on the glycosaminoglycans. As Fig. 5 shows, staining of hyaluronate. which has only carboxyl groups, is highly dependent on the concentration of acetic acid (absorbance de-

GLYCOSAMINOGLYCAN

creases from 0.658 in the absence of acetic acid to 0.253 in the presence of 0.875 M acetic acid). In contrast, staining of keratan sulfate-l, which has only sulfate groups and no hexuronic acid, is unaffected by increasing the acetic acid concentration (Fig. 5). Heparin and chondroitin 6-sulfate, which have both carboxyl and sulfate groups, show an intermediate response to increasing acetic acid concentrations. The effect of acetic on dye uptake is modified by the MgC& concentration. In the absence of MgCl, the effect of acetic acid on dye uptake by all glycosaminoglycans, including hyaluronate, is very small (data not shown). In the case of keratan sulfates, if the MgCI, concentration is increased above 0.100 M a significant decrease in dye uptake is observed when the acetic acid concentration is also increased to 0.875 M. This effect becomes more pronounced as the MgCl, concentration of the dye and rinse solutions is increased (data not shown). Efjkct of Solvent Polarity and Temperature on the Staining Intensity of the Glycosaminoglycans

Table 4 summarizes data on dye uptake by the glycosaminoglycans using different staining methods at two different temperatures. Dye uptake is increased by up to 7% at 5.5 compared to 23°C in stain I and up to 12% at the lower temperature in stain II method. The only glycosaminoglycan that appears to deviate from this trend is chondroitin 6-sulfate and the differences here are less than 3%. Staining intensity of the glycosaminoglycans by stain II (which contains five times the ethanol concentration of stain I) is lower for all glycosaminoglycans except the keratan sulfates. compared to stain I method. The greatest difference is observed for the hyaluronate which stains only about 40% as intensely in stain II method as in stain I method. Stain III method differs from stain I

401

CHARACTERIZATION TABLE EFFECT

OF TEMPERATURE DIFFERENT

Stain GAG Hya ChCS Ch6-S DeS HeS Hep KeS- I KeS-2

4

5.X 0.865 0.805 0.802 0.754 0.840 0.885 0.598 0.566

ON DYE

STAINING

I

UPTAKE

IN

SOLUTIONS

Stain

II

Stain III

23°C

5.5”C

23°C

23°C

0.803 0.772 0.824 0.715 0.811 0.830 0.550 0.547

0.355 0.698 0.798 0.606 0.635 0.803 0.676 0.662

0.319 0.623 0.821 0.554 0.632 0.783 0.594 0.618

0.504 0.470 0.543 0.474 0.605 0.597 0.284 0.323

Note. Stain I contains 0.2% Alcian blue (w/v). 10% ethanol (v/v), 0.030 M MgCl,, and 0.1% acetic acid (v/v). Stain II contains 0.2% Alcian blue (w/v). 504 ethanol (v/v), 0.030 M MgCl,, and 0.1% acetic acid (v/v). Rinse solutions for stains 1 and II are identical with the staining solutions. but without the Alcian blue. Stain III contained 0.2% Alcian blue (w/v), 10% ethanol (v/v), and 5% acetic acid (v/v). Rinse for stain 111 was 5% acetic acid (v/v). Staining time in all stain solution\ was 30 min. Constant amount of each glycosaminoglycan was used for all three stains. Absorbance was measured at 677 nm in DMSO containing 0.094 M HLSO,.

method in containing 5% (v/v) acetic acid instead of 0.1% (v/v) and in lacking MgCl,. It is seen that dye uptake by the glycosaminoglycans in stain III method is considerably less than that in the stain I method. This difference is especially marked for the keratan sulfates, which are highly dependent on MgCI, for maximal dye uptake, and where an increase from 0 to 0.030 M MgCI, increased dye uptake by approximately twofold (Fig. 1). Similarly, for the other glycosaminoglycans the decrease is due primarily to the absence of MgCl,, rather than to the presence of the additional acetic acid (see previous section on effect of acetic acid).

Table 5 summarizes data on the specificity of these enzymes. Chondroitinase treat-

402

HRONOWSKI TABLE

QUANI ITATIVE GLY~OSAMINOGLYCANS LJRONIDASE

AND

ASSAY

AND

5

OF SMALL AMOUNTS OF USING TESTICULAR HYAI.-

CHONDROITINASE

Chondroitinase ABC

ABC

ANASTASSIADES

4 and 10% after 2 h of treatment (data not shown). Absorbance by stained dermatan sulfate, heparin, heparan sulfate, and the keratan sulfates was not significantly affected by hyaluronidase treatment.

Hyaluronidase

GAG

Control

Enzyme treated

Control

Enzyme treated

Hya Ch4-S Ch6-S DeS HeS b KeS-1 KeS-2

0.817 0.747 0.811 0.675 0.793 0.828 0.558 0.530

0.665 0 0 0.006 0.786 0.820 0.488 0.506

0.808 0.814 0.850 0.670 0.745 0.729 0.580 0.460

0 0.017 0.011 0.656 0.743 0.724 0.575 0.447

Nole. Samples (50 ~1) of glycosaminoglycan were treated with the enzymes as described under Materials and Methods. Controls were treated identically to the enzyme-treated samples, with the exception that they lacked the enzymes. Samples were lyophilized and redissolved in 50 ~1 of water, and stained as described under Materials and Methods. Absorbance was measured in 2 ml of DMSO containing 0.094 M H,SO, at 677 nm.

ment completely prevented the staining of the chondroitin sulfates. In the case of dermatan sulfate approximately 1% of the absorbance remained after 30 min of incubation with chondroitinase. Hyaluronate is also partially digested by the chondroitinase, but heparin and heparin sulfates are not significantly affected. The difference in absorbances between control and chondroitinase-treated samples of keratan sulfate is also not considered significant, since in a similar experiment with longer incubation times there was only a difference in absorbances of approximately 1%. Hyaluronidase completely prevented staining of hyaluronate. and staining is completely prevented even after 2 h of treatment by the procedure described (Materials and Methods). Chondroitin 6-sulfate and chondroitin 4-sulfate yield only 1 and 2’95, respectively, of the original absorbance after 8 h of hyaluronidase treatment (Table 5) and

In addition to the glycosaminoglycans, Alcian blue will stain nucleic acids and certain glycoproteins. In order to determine the specificity of the present staining procedure various macromolecules were tested (Table 6). With the present procedure the staining intensity of nucleic acids is considerably less than that of the glycosaminoglycans. On a per unit weight basis DNA stains only about 14% as intensely as the glycosaminoglycans. while RNA. only about 4% intensely. In addition, the behavior of DNA and RNA on cellulose acetate electrophoresis is considerably different from that of the glycosaminoglycans. DNA does not migrate in the electrophoresis system used here and most of the RNA also remains at the origin, while a small component migrates slowly relative to the glycosaminoglycans, as a diffuse band. The extent of staining of the glycoproteins is very small, compared to that of the glycosaminoglycans. On a per unit weight TABLE ALCIAN BI.UE STAINING ACIDS AND GLYCOPROIEINS CONTAINING~.?~ AILIAN (v/v), 0.030 M M&I,, AND

6 Irvlt:NsIrY IN

Amount (/l!z)

Pepsin Phytohemagglutinin Fetuin

7.5 19.2 24.8 19.5 23.0

DNA

M

OF

NUCI.EIC SOLUJ.ION

BLUE (w/v). 10% ETHANOI 0. I% ACE I IC ACID (v/v)

Substance

RNA

S-IAIN

Absorbance 0.291 0.217 0.056 0.006 0

Note. The procedure for staining these substance> was exactly the same as that used for the glycosaminoglycans. Absorbances were measured in I.5 ml of DMSO containing 0.094 %I H,SO, at 677 nm.

GLYCOSAMINOGLYCAN

basis dye uptake by pepsin is less than 1% and by phytohemaglutinin 0.1% compared to that of the glycosaminoglycans. No staining by the present procedure was observed for fetuin, trypsin, trypsin inhibitor, hyaluronidase, protease, acid phosphatase, papain, Pronase, concanavalin A, and albumin. By increasing the concentration of the MgCl, in the stain from 0.030 to 0.10 M, dye uptake by pepsin was reduced by 80% and by the nucleic acids by 40%. DISCUSSION

The method described for characterizing small amounts of glycosaminoglycans involves selective elution of the glycosaminoglycans after they have been separated on cellulose acetate, although electrophoretic separation is not an essential step in characterization by elution. It has been pointed out that this method is especially useful for distinguishing between dermatan sulfate and heparan sulfate, which general1 y migrate close together in electrophoretic systems. However. unlike enzymatic and chemical methods, which degrade glycosaminoglycans the present elution method gives useful information about the intact polymer from the elution profile. It has been shown (19) that the salt concentration at which a particular glycosaminoglycan-organic cation complex becomes soluble is dependent on the valency of the polyanion, which is in turn proportional to the molecular weight of the polymer. This predicted pattern of elution is observed with the chondroitin sulfates in Fig. 1, where chondroitin 6-sulfate, which has a viscosity average molecular weight of 29,000 and 0.90 sulfate esters per hexosamine group, requires higher MgCl, concentration for elution than chondroitin 4-sulfate. which has a viscosity average molecular weight of 15,000 and 0.98 sulfate esters per hexosamine residue. However, the elution pattern is also dependent on the source of the material (Fig. 2) where both the type of sulfation and

CHARACTERIZATION

403

molecular weight can vary (32). Chondroitin sulfate reference standards from the river sturgeon elute off at lower MgC& concentrations than the homologous commercial preparations from the shark and the whale as would be expected for a greater linear density of charged groups. Chondroitin sulfate synthesized by rat muscle cells elutes off at an intermediate salt concentration (Fig. 3). Separation of the sulfated glycosaminoglycans by electrophoresis is generally not complete. In the present electrophoresis system the chondroitin sulfates overlap with the keratan sulfates, but migrate faster than dermatan sulfate and slower than heparin. Enzymatic methods in the sensitivity range of the staining procedure have been described, but enzymes like hyaluronidase and chondroitinase ABC strongly bind to certain glycosaminoglyans such as heparan sulfate, and when the enzyme digest is subjected to cellulose acetate electrophoresis these bound complexes remain at the origin. The elution method does not suffer from this disadvantage and in addition allows separation, quantitation, and partial characterization of the sample on cellulose acetate, without the need of transfer of the sample, which may be in the low microgram scale. In order to gain insight as to the nature of the dye-polyanion interactions, it should be noted that Alcian blue is a molecule consisting of large nonpolar copper phthalocyanin moiety with an average of approximately four tetramethylisothiouronium groups attached at the periphery of the ring, to make the dye soluble in aqueous solvents. The structure of Alcian blue has been extensively investigated (21) and in the subsequent discussion it will be assumed that there are four S-methylene tetramethylisothiouronium side chains per phthalocyanin molecule. Removal of the nonpolar regions of the dye from an aqueous environment would be expected to lead to an increase in entropy and thus to a decrease in the free energy of the system.

404

HRONOWSKI

AND

The solubilization of the glycosaminoglycan Alcian blue complex has been previously (19,20) discussed in terms of the polarizability of the interacting ions. This explains the large difference in MgCl, concentration needed to elute hyaluronate, and most of the sulfated glycosaminoglycans (Fig. 1). However, Fig. 1 also illustrates that dye uptake by the sulfated glycosaminoglycans is still increasing as the concentration of MgCl, is increased beyond that required to elute all of the hyaluronate off the strip (0.1 M MgCl,). In addition, the moles of dye bound by glycosaminoglycans containing both sulfate and carboxyl groups (except heparin) exceeds the molar sulfate content by 35-63% (Fig. 1 and Table 3). In the case of the keratan sulfates the excess is between 8 and 16%. Heparin under maximal uptake conditions binds on the average only 6.3 Alcian blue molecules per 10 sulfate ester groups. The lower amount of dye bound by heparin per sulfate group may be due to steric hindrance or may be due to elution of the smaller fragments of heparin so that, at the MgCI, concentration giving maximal dye uptake, only a fraction of the original heparin sample is stained. The above observations are consistent with the notion that entropy effects are important in stabilizing the dye-glycosaminoglycan complexes. The data in Table 4 give further support to this view. As the ethanol concentration in the stain method is increased to 50% (v/v), thus making the solvent less polar and also decreasing the contribution of entropy to the stabilization of the complexes, staining intensity is reduced for all of the glycosaminoglycans, except for the keratan sulfates, which stain more intensely at the higher ethanol concentrations. The maximal decrease in dye uptake was observed for hyaluronate in the presence of 50% ethanol. it should be noted, however, that stain I and stain II contained 0.030 M MgCl,. which is below the optimal concentration for maximal dye uptake by the sulfated glycosaminoglycans and that

ANASTASSIADES

even the increased dye uptake in stain II by the keratan sulfates is still less than the expected maximum on the basis of the number of sulfate ester groups. The observed changes in dye uptake by hyaluronate and the keratan sulfates in stain I and stain II solutions suggest that two processes may be taking place. At the higher ethanol concentration there is likely increased dye binding to the sulfate groups and decreased dye binding by the carboxyl groups, as the stabilization of adjacent dye molecules due to entropy effects is reduced. The net decrease in dye uptake in most of the sulfated glycosaminoglycans under less polar conditions, is thus consistent with a large decrease in dye binding by the carboxyl groups and a smaller increase in dye binding by the sulfate groups. From these considerations a dye-glyccosaminoglycan complex is envisaged with the dye molecules stacked on one side and anchored to the polyanion by salt links. The excess dye bound over the sulfate content may then be stabilized on the dye-glycosaminoglyan complexes by hydrophobic interactions with adjacent dye molecules. It can be seen from the Results section (Fig. 1 and Table 2) that the presence of salt in the solvent in contact with the stained strips is essential for retention of maximal dye uptake by the glycosaminoglycans. The results with hyaluronate show that the elution is not complete in the stain, solution, but occurs also during the rinse steps. The results with heparin suggest that some heparin elutes off the strip even before the maximal absorbance plateau is past, suggesting that further dye uptake occurs by a population of the heparin molecules while other, probably smaller fragments, are eluting off the strip. The net effect is that of constant absorbance, which could be erroneously interpreted as no elution in the plateau region (Fig. I). An important practical consideration of the high correlation coefficient of dye uptake with the weight of the sulfated glycosaminoglycans when stained in the presence of

GLYCOSAMINOGLYCAN

0.200 M MgCl, (Table 3) is that only a single standard curve is needed to quantitate the sulfated glycosaminoglycans at this MgCI, concentration. The method is quantitative with a practical lower limit of 0.1 pg of material. ACKNOWLEDGMENT This Council

work was supported by the Medical of Canada. Grant MT-3603.

Research

REFERENCES 1. Hronowski, L., and Anastassiades, T. P. (1979) Anul. Biorhrm. 93,60-72. 2. Breen, M., Weinstein. H. G., Black. L. .I.. Borcherding, M. S.. and Sittig. R. A. (1976) in MethodsofCarbohydrate Chemistry (Whistler. R. L.. and BeMiller, J. N.. eds.), Vol. 7. pp. 101-115. Academic Press, New York. 3. Hata. R., and Nagai. Y. (1972) Anal. Biochrm. 45, 462-468. 4. Stefanovich, V.. and Gore. I. (1967) J. Chromatogr. 31,473-478. 5. Seno, N., Anno, K., Kondo, K., Nagase. S.. and Saito, S. (1970) Anal. Biwhem. 37, 197-202. 6. Hsu, D.. Hoffman, P., and Mashburn. T. A., Jr. (1972) Anal. Biochem. 46, 156- 163. 7. Curwin. K. 0.. and Smith. S. C. (1977) Arm/. Biochem. 79, 291-301. 8. Inoue. S., and Iwasaki. M. (1976) J. Bioc,hrm. 80, 513-524. 9. Saito, H., Yamagata, T.. and Suzuki, S. (1968) J. Biol. Chem. 243, 1536-1542. 10. Scott. J. E.. and Dorling. J. (I%S) Hi.stochemir 5, 221-233. 11. Whiteman, P. (1973) B&hem. J. 131, 343-350. 12. Bitter, T.. and Muir, H. (1962) Awl. Biochrm. 4,330-334. 13. Ainsworth, T., Puzic. 0.. and Anastassiades. 0. (1977) J. Lab. C/in. Med. 89, 781-791.

CHARACTERIZATION

405

14. Castor, C. W., Wright, D., and Buckingham. R. B. (1968) Arthriri.r Rheum. 11, 652-659. 15. Roden. L., Baker, J. R., Cifonelli, .I. A., and Mathews. M. B. (1972);~ Methods in Enzymology (Ginsburg V.. ed.), Vol. 28, pp. 73- 140, Academic Press, New York/London. 16. Dulbecco, R., and Vogt. M. (1954) J. Erp. Met/. 99, 167- 182. 17. Taylor. R. L.. Shively, J. E., Conrad, H. E.. and Cifonelli. J. .4. (1973) Biochemistp 12. 36333637. 18. Chatfield, C. (1975) Statistics for Technology. Chapman & Hall. London. 19. Scott. J. E. (1970) itz Chemistry and Biology of the Intercellular Matrix (Balays, E. A., ed.), Vol. 2. pp. 1105-1119, Academic Press. London. 20. Bungenberg de Jong. H. (1949) in Colloid Science (Kuyt, H. R., ed.). Elsevier. Amsterdam. 21. Scott, J. E. (1972) Hisiochrmir 30, 215-234. 22. Dische. Z. (1947) J. Biol. Chum. 167. 189- 198. 23. Elson, L. A.. and Morgan, W. T. J. (1933) Biw them. J. 27, 1824- 1828. 24. Dodgson, K. S., and Spencer, B. (1953) Biochum. J. 55, 436-440. 25. Muir, H. (1958) Biochem. J. 69, 195-204. 26. Cifonelli. J. A.. and Dorfman, A. (1960) J. Biol. C‘hem. 235, 3283-3286. 27. Cifonehi. J. A., and King, J. ( 1970) Curhohyd. Res. 12, 391-402. 28. Dische. Z. ( 1955) Mrfhods Biochrm. Anal. 2, 313-358. 29. Roden, L.. Baker, J. R., Cifonelli, J. A., and Mathews, M. 8. (1972) in Methods in Enzymology (Ginsburg. V., ed.), Vol. 28, Part 9, pp. 131-135. Academic Press. New York. 30. Mathews, M. B. (1975) Connective Tissue: Macromolecular Structure and Evolution. Table 10.1 p. 215. Springer-Verlag, New York. 31. Mathews. M. B. (1%7) Biol. Re\,. 42, 499-551. 32. Mathews. M. B. (1975) Connective Tissue Macromolecular Structure and Evolution, Chaps. 6-8. Springer-Verlag. New York.