Cationic heme undecapeptide as stain for detecting glycosaminoglycans on cellulose acetate strips

ANALYTICAL

BKICHEMISTRY

155,78-82 (1986)

Cationic Heme Undecapeptide as Stain for Detecting Glycosaminoglycans on Cellulose Acetate Strips’ NICOLAE

GHINEA

Institute of Cellular Biology and Pathology, Bucharest-79691, Romania Received October 10, 1985 A rapid and sensitive method has been developed for detecting glycosaminoglycans on cellulose acetate strips. The method is based on the electrostatic interactions between glycosaminoglycans and cationic heme undecapeptide. The complexes are identified by peroxidatic reaction using 3J’diaminobenzidine asHz donor. The detection limit is 2.5-10 ng for sulfated glycosaminoglycans and 60 ng for hyaluronic acid. The sensitivity of this method is higher than of those using Alcian blue or toluidine blue (500 ng). 0 1986 Academic press Inc. KEY WORDS: glycosaminoglycans; electrophoresis; cationic heme undecapeptide.

hhhGlycosaminoglycans (GAG)* are hhigh-tion between GAG and cationic heme unmolecular-weight polyanionic heteropolysacdecapeptide (Fig. 1) and subsequent staining charides which, with the exception of hyal- of these complexes by taking advantage of the uranic acid, are covalently bound to a core peroxidatic activity of the latter. protein (proteoglycans). All glycosaminoglycans have acid sulfate and/or carboxyl groups MATERIALS AND METHODS and many of their functional properties have been attributed to their polyelectrolyte nature Microperoxidase M- 11 (heme undecapep(l-3). Their high negative charge has been tide), I -ethyl-3(3-dimethylaminopropyl) carused to advantage for their separation from bodiimide hydrochloride, papain (Type IV, 2 crude mixture and for further characterization. X crystallized), and 3,3’-diaminobenzidine The method most widely employed for the tetrahydrochloride were purchased from identification of these compounds is electro- Sigma Chemical Company (St. Louis, MO.), phoresis on cellulose acetate strips and agarose cellulose acetate electrophoresis strips (Segels followed by staining with cationic dyes praphore III) from Gelman Sciences Inc. (Ann (4-l 1). When stained with Alcian blue and Arbor, Mich.), and pyridine, formic acid, hytoluidine blue, the detection limit of GAGS is drogen peroxide, cadmium acetate, cupric acabout 500 ng (4,ll). etate, barium acetate, calcium acetate, Pronase This report presents a general staining E, and NJ-dimethyltrimethylenediamine method for detecting GAGS on electrophofrom Merck-Schuchardt (Darmstadt, FRG). retograms based on the electrostatic interacCationic heme undecapeptide was prepared in our laboratory from microperoxidase M- 11 by covalently blocking the free carboxyl groups ’ This work was supported by the Ministry of Education, with N,N-dimethyltrimethylenediamine ( 12). Romania, and by NIH Grant HL-26343 awarded to Nicolae Simionescu and Maya Simionescu. The following protocol was used: 25 mg of ’ Abbreviations used: GAG, glycosaminoglycan; C4S, HUP was dissolved in 2.5 ml of 2 M N,Nchondroitin 4-sulfate; C6S, chondroitin 6-sulfate; HS, he(pH 4) with paran sulfate; HP, heparin; HA, hyaluronic acid; DS, der- dimethyltrimethylenediamine continuous stirring. After HUP solubilization matan sulfate; KS, keratan sulfate; HUP, heme undeca100 mg 1-ethyl-3( 3-dimethylaminopropyl) peptide; cHUP, cationic heme undecapeptide. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

78

PEROXIDATIC

STAINING

OF GLYCOSAMINOGLYCANS

79

Preparation of crude GAGS. As a starting material for the detection of GAGS from tissue extracts, rabbit tracheal cartilage was used. The specimens of cartilage were cut into small pieces, dehydrated with several changes of acetone, and then completely delipidated with CHC13:CH30H (2: 1). The weight dry cartilage was processed for proteolytic digestion by using papain and pronase E (10). After a total digestion of 52 h and addition of trichloroacetic acid to a final concentration of 5%, the mixture was kept cold for a few hours. The precipitate was removed by centrifugation and the solution was neutralized, dialyzed against (CHj2 several changes of distilled water, and finally co lyophilized. Uranic acid was determined by the carbazol method of Bitter and Muir (13) with glucuronic acid as standard. One-dimensional electrophoresis. Electrophoreses were carried out in six different solvent systems: (a) 0.1 M pyridine/0.47 M formic acid (pH 3), constant current 1 mA/cm for 30 FIG. I. Structural formulas of cationic heme undecamin; (b) 0.1 M cadmium acetate (pH 4), conpeptide (cHUP), the cationic dye with peroxidatic activity stant current 0.5 mA/cm for 1.5 h; (c) 0.1 M used for the identification of GAGs on electrophoretocupric acetate (pH 5.3) constant current 0.5 grams. mA/cm for 1.5 h; (d) 0.1 M calcium acetate (pH 7.2) constant current 1 mA/cm for 3 h; carbodiimide hydrochloride was added and (e) 0.1 M barium acetate (pH 8), constant curthe pH was maintained at 4 with 0.2 N HCl rent 1 mA/cm for 3 h; (f) 0.1 M N,N-dimethuntil no detectable changes in pH were re- yltrimethylenediamine (pH 8.5) constant curcorded (- 3 h). The reaction mixture was left rent 0.5 mA/cm for 1 h. for 48 h at 22°C and then passed through a Cellulose acetate strips were dipped in the Sephadex G-25 fine column (90 X 1.5 cm) solvent system and lightly blotted with a filter with distilled water as eluant. cHUP was con- paper. In order to obtain the detection limits centrated by lyophilization (Multidry, FIS by peroxidatic staining of GAGS, 1 ~1of either System Inc., Stone Ridge, N.Y.). The dye so- serial dilutions of each GAG or of an authentic lution was prepared by dissolving cHUP pow- mixture of them, were applied to the plates at der in 0.075 M NaCl at concentration of 0.1% 10 mm from the cathode end. For the iden(w/v) and the pH of the solution was adjusted tification of the rabbit tracheal cartilage GAGS to 7 with 0.1 N NaOH. This solution, stored we used coelectrophoresis with one or a mixat 4°C was successfully used in 100 staining ture of standards. The electrophoreses were steps. performed at a constant voltage of 180 V at Standards of glycosaminoglycans (heparin, 10°C using a Desaphor HM device (Desaga, chondroitin 4-sulfate, chondroitin 6-sulfate, Heidelberg, FRG). dermatan sulfate, heparan sulfate, keratan Two-dimensional electrophoresis. Cellulose sulfate, hyaluronic acid) have been kindly acetate strips kept overnight in 30% (v/v) provided by M. B. Mathews and J. A. Chifomethanol were dipped in 0.1 M pyridine/0.47 nelli. M formic acid buffer, pH 3, for 30 min, lightly NW32

80

NICOLAE

c4s KS HA

q<,

HS HP

:

1

21

2

FIG. 2. Coelectrophoretic separation of rabbit tracheal cartilage GAGS (1) with a mixture of five standards (2) on cellulose acetate strips. Electrophoresis was performed at I80 V (1 mA/cm for 3 h using 0.1 M barium acetate, pH 8, as an electrolyte. cHUP was used as a cationic dye with peroxidatic activity. The strips were incubated in 0.025% 3,3’-diaminobenzidine and 0.02% Hz02 in 0.02 M TrisHC1 buffer, pH 8.1. Observe that the tissue extract contains traces of a HP-like compound.

blotted, and equilibrated for few minutes before sample application (4). Aliquots of 2 ~1 of a glycosaminoglycans mixture (equivalent to 100 ng in each compound) were applied in drops at the origin (10 mm from the cathode end for the first run, and 20 mm from the cathode side for the second). Electrophoresis in the first dimension was carried out at 10°C

GHINEA

in 0.1 M pyridinef0.47 M formic acid, pH 3, at 1 mA/cm for 30 min, and in the second dimension, in 0.1 M barium acetate, pH 8, at 1 mA/cm for 2 h. Staining. After electrophoresis, the strips were briefly washed with deionized water and immersed in 0.1% cationic heme undecapeptide in 0.075 M NaCl, pH 7, for 10 min. The unbound cationic heme undecapeptide was removed by three successive washings with a solution of 0.075 M NaCl(3 min); then strips were incubated in 0.025% 3,3’-diaminobenzidine and 0.02% H202 in 0.02 M Tris-HCl buffer, pH 8.1 ( 10 min). At longer periods of time (up to 48 h) a better contrast was obtained. After washing with water, the electrophoretograms were stored. RESULTS

The coelectrophoreses with one or more standard GAGS indicated the presence of C4S, KS, HA, and traces of HP in the rabbit tracheal cartilage. A pattern of GAG distribution obtained by peroxidatic staining using cHUP as a cationic dye is illustrated in Fig. 2. A high content of C4S vs KS from cartilage tissue extract can be observed. In order to test the influence of the electrophoretic buffer system on the staining step, six solvent systems have been used (see Materials

TABLE 1 DETECTION

LIMITS

OBTAINED BY PEROXIDATIC STAINING OF GLYCOSAMINOGLYCANS ELECTROPHORESIS ON CELLULOSE ACETATE STRIFE

Glycosaminoglycan

Source

Hyaluronic acid Chondroitin 6-sulfate Chondroitin 4-sulfate Dermatan sulfate Heparin Heparan sulfate Keratan sulfate

Human umbilical cord Sturgeon cranial cartilage Sturgeon notochord Hog mucosal tissue Beef lung Beef lung Human costal cartilage

Molecular weight” 230,000 29,000 15,000 45,000

14,000 -

Degree of sulfation” 0 0.90 0.98 1.13 2.77 0.97

1.26

AFTER

Detection limit (w/d) 60 10 IO 5 2.5 IO 5

u The molecular weights and degrees of sulfation were determined by Dr. M. B. Mathews and Dr. J. A. Chifonelli (University of Chicago) and were mentioned in the sheet accompanying the samples.

PEROXIDATIC

STAINING

and Methods). Irrespective of the electrophoretie solvent system, using serial dilutions of each standard GAG (starting from 0.5 pg/& the detection limits obtained by the peroxidatic reaction of the GAG/cHUP complexes were 2.5 ng for HP, 5 ng for DS and KS2, 10 ng for C6S, C4S and HS, and 60 ng for HA (Table 1). Irrespective of the electrophoretic solvent system, when serial dilutions of a mixture of standard GAGS were used, similar results were obtained but the GAGS were not satisfactorily separated from one another (Fig. 3). This was achieved by the two-dimensional electrophoresis technique (Fig. 4). DISCUSSION

In order to improve the detection limits of electrophoretically separated GAGS, we developed a new staining method by using cationic heme undecapeptide as a cationic dye. Cationic heme undecapeptide is a small heme peptide (&I, 2215) with high positive charge (PI 9) ( 12) which tightly binds by electrostatic forces to polyanionic GAGS. Due to the amplifying effect of its peroxidatic activity, cationic heme undecapeptide can generate a large amount of reaction product by the oxidation of 3,3’-diaminobenzidine (HZ donor)

HP

e

e

500

250

120

60

P

-

30

10

_ 5

1 2,5ng

FIG. 3. Electrophoretic separation of an authentic mixture of five standard glycosaminoglycans on cellulose acetate strips. The mixture (equivalent to 0.5 pg in each compound) was serially diluted and 1 ~1 of each dilution was applied to the strip. Electrophoresis was performed at 180 V (1 mA/cm) for 3 h using 0.1 M barium acetate, pH 8, as an electrolyte. After electrostatic interaction with cationic heme undecapeptide, the glycosaminoglycan/cationic heme undecapeptide complexes were identified by peroxidatic reaction using 3J’diaminobenzidine as H2 donor and HzOz as substrate. The detection limits are marked with arrowheads.

81

OF GLYCOSAMINOGLYCANS

HA

5 origm ls'

ties electrophoresls

3)HP L

FIG. 4. Peroxidatic staining with cationic heme undecapeptide after two-dimensional electrophoresis of an authentic mixture of standard glycosaminoglycans. Two microliters of solution (equivalent to 100 ng in each compound) were applied in drops at the origin and the electrophoresis was then carried out in 0.1 M pyridine/ 0.47 M formic acid, pH 3, at 1 mA/cm for 30 min in the first dimension, and in 0.1 M barium acetate, pH 8. at 1 mA/cm, for 2 h in the second dimension.

with hydrogen peroxide (substrate) (12). As a result, the sensitivity of GAG detection is markedly increased. Depending on the GAG, the detection limit was 2.5-10 ng for the sulfated compounds and 60 ng for hyaluronic acid (Table 1). The sensitivity of this method appears to be markedly higher than that reported for Alcian blue (4,7,1 l), toluidine blue (9,l I), high-iron diamine (14), or the indirect staining method using serum albumin (15). It has been demonstrated (16) that the interaction between GAGS and cationic polypeptides depends on: (a) the position of the carboxyl groups, (b) the glycosidic linkages, (c) the position of the sulfate groups, and (d) the degree of sulfation. These factors operate in our system as well, and can account for the difference observed between the detection limits of different GAGS. Good results were obtained when this method was applied to identification of GAGS from crude extracts that had not been as extensively purified as the standards used in the present study. We demonstrate that the rabbit tracheal cartilage

82

NICOLAE

is particularly rich in chondroitin 4-sulfate and contains small amounts of keratan sulfate, hyaluronic acid, and, unlike other cartilage tissues, traces of heparin-like compounds are detected (Fig. 2). The method described here can be adapted for quantitative analysis of GAGS on electrophoretograms as well as in samples by using nitrocellulose membranes or Linbro plates as carriers of GAGS (manuscript in preparation). ACKNOWLEDGMENTS Expert technical assistancewas provided by M. Toader, V. Craciun (biochemistry), V. G. Ionescu and E. Stefan (photography), and M. Mazilu (secretarial work). REFERENCES

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