Quantitation of Proteoglycans in Biological Fluids Using Alcian Blue

Madeleine Karlsson and Sven Bjornsson 1. Introduction

Alcian blue is a tetravalent cation with a hydrophobic core that contains a copper atom, hence its blue color (1). The four charges allow Alcian Blue to bind to gly-cosaminoglycans (GAGs) at high ionic strength, in contrast to other cationic dyes, which are all monovalent. The molecular structure, that is, the plane tetragonal hydro-phobic core with positive charges attached at its corners, may facilitate formation of aggregates of several molecules side by side rather than micelle formation. The ionic strength and presence of other detergents will affect the size of these aggregates in solution. Such positively charged linear aggregates will bind strongly to some negatively charged polymers such as GAGs, but not to others such as nucleic acids reflecting the conformation of both the polymer and the dye. Alcian blue is not a well defined substance. There are large differences in solubility and binding characteristics among different manufacturers but also between batches from the same manufacturer. In addition, the stain has a limited shelf life and is slowly degraded both in solution and as dry substance.

Glycosaminoglycans are negatively charged polymers built by repeating units of disaccharides containing one uronic acid or galactose and one N-acetylglycosamine. The variation in charge may be very large, since each disaccharide is more or less sulfated. The ionic bonding between cationic dyes and the negatively charged GAGs is generally thought to be proportional to the number of negative charges present on the GAG chain—that is, both sulfate and carboxyl groups. In contrast, studies of the interaction at different pH and ionic strength (2) revealed that the carboxyl groups are not involved in the binding of Alcian blue to GAGs. Moreover, the presence of charged carboxyl groups interferes with binding of Alcian blue to the GAGs. One species of GAG, keratan sulfate, contains not uronic acid but galactose, and thus lacks the

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

carboxyl group. Accordingly, the binding of Alcian blue to keratan sulfate is not pH dependent, and the amount of dye bound per milligram keratan sulfate is equal to that of other types of GAGs (2).

The principle is based on the specific interaction between sulfated polymers and the tetravalent cationic dye Alcian blue at a pH low enough to neutralize all carboxylic and phosphoric acid groups and at an ionic strength great enough to eliminate ionic interactions other than those between Alcian blue and sulfated GAGs (2). Hyaluronan, a nonsulfated GAG, does not react in this assay. There is no interference from proteins or nucleic acids in this method (see Figs 1 and 9, later, and refs. 2 and 4), in contrast to the dimethylmethyl blue (DMMB)-method (5,6) or other dye-binding methods. At 0.4 M guanidine-HCl, binding of Alcian blue to GAG chains in proteoglycans is the same as binding to isolated GAG chains. Thus, the method is equally applicable to proteoglycans samples as to GAG samples.

A method for simultaneous quantitation and preparation of intact proteoglycans in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published (2). The tube/absorbance assay has a measuring range of 1-20 ^g of GAG. Proteoglycans prepared by Alcian blue precipitation are suitable for electrophoresis (3) or biochemical analysis. The assay principle can be applied at any scale, from a 50 ^L sample in a quantitative assay to a 600-mL sample in large-scale preparation.

Quantitation may also be performed using the dot blot format. In the dot blot/reflectance assay, the Alcian blue-GAG complexes are collected on a polyvinylidene diflouride (PVDF)-membrane by filtration in a dot-blot apparatus, and the stain is quantitated as reflectance by scanning densitometry. The dot-blot/reflectance assay has a measuring range of 10-800 ng of GAG, which is enough to measure the low contents of proteoglycans in plasma, urine, or wound fluid.

2. Materials

1. Alcian blue, certified for GAG/PG quantitation, Wieslab, Lund, Sweden.

2. Chondroitin-6-sulphate (C-4384) from shark cartilage, keratan sulphate (K3001) from bovine cornea, heparan sulphate (H7640) from bovine kidney, from Sigma (USA).

3. Capped polypropylene vials (1- or 2-mL), Sarstedt, Sweden.

4. Centrifuge with a fixed-angle rotor for 2-mL vials, and a minimal centrifugal force 12,000g.

5. ELISA photometer equipped with a 605- or 620-nm filter.

6. Syringe (5-10 mL) with a large-bore needle (1.5 mm).

7. MilliBlotD system (#MBBDDD 960) dot-blot apparatus, Biogenex, San Ramon, CA, USA.

8. PVDF membrane (Immobilon-P #IVPH00010), Millipore, USA.

9. A flat-bed scanner, Scanmaker II (Mikrotek Lab, USA) and a PowerMacintosh with Adobe Photoshop™ (Adobe Systems, USA) was used for imageing.

2.1. Stock Solutions

1. 10% (v/v) H2SO4: Add 50 mL of H2SO4 slowly with stirring to 450 mL of H2O to a final volume of 500 mL. The solution will be stable for for 1 yr at room temperature.

2. 10% (w/v) Triton X-100 : Dissolve 50g of Triton X-100 in 500 mL of H2O, stir until in solution. The solution will be stable for for 1 yr at room temperature.

3. Alcian blue stock solution (Alcian blue in 0.1% H2SO4/0.4 M guanidine-HCl ): Add 0.1 mL of 10% H2SO4 to 0.5 mL of 8 M guanidine-HCl. Add H2O to 10 mL. Dissolve 0.1-0.2g (depending on batch) of Alcian blue 8 GS (Wieslab, Sweden). Mix for 2 h to overnight. Store in the dark at 4°C. The solution will be stable for for 1 mo.

The guanidine-insoluble portion of the stain is removed by centrifugation (12,000g, 15 min) before each preparation of AB reagent. Dilute 100 ^L of the supernatant to 10 mL and read absorbance at 600 nm. The concentration of Alcian blue in the stock solution should be adjusted, by addition of 0.4 M guanidine-HCl/0.1% H2SO4, corresponding to an absorbance of 1.4 of the diluted stock.

4. 8.0 M guanidine-HCl (Gu-HCl): Dissolve 764 g of guanidine-HCl p.a. (Mw 95.53) in H2O and add to 1000 mL. The solution will be stable for for 1 yr at room temperature. If technical-grade guanidine-HCl is used, it must be stirred overnight with active charcoal and then filtered. The concentration of a guanidine-HCl may be checked by measuring the density of the solution: 8.0 M guanidine-HCl has a density of 1.192 g/L.

5. 3.0 M MgCl2: Dissolve 305 g of MgCl2 x 6H2O (Mw 203.30) in H2O and add H2O to 500 mL.The solution will be stable for for 1 yr at room temperature.

6. CsC calibrator stock solution (chondroitin-6-sulfate 10mg/mL): Dissolve 20 mg of chon-droitin-6-sulfate (Sigma C4384) in 2 mL of H2O. The solution will be stable for several years at -20°C.

7. TG x 10 (0.25 M Tris/1.92 M glycine pH 8.3): Dissolve 12.1 g Tris base and 57.6g of glycine in a final volume of 400 mL of H2O.The pH should be approximately 8.3, but need not be adjusted. The solution will be stable for for 1 yr at room temperature.

8. 10% (w/v) SDS: Dissolve 25 g of SDS in a final volume of 250 mL of H2O. The solution will be stable for for 1 yr at room temperature.

9. 1.0M Tris-acetate, pH 7.4: Dissolve 121 g of Tris base in H2O. Adjust pH to 7.4 with acetic acid. Add H2O to 1000 mL. The solution will be stable for for 1 yr at room temperature.

2.2. Reagents

1. SAT-reagent (0.3% H2SO4/0.75% Triton X-100): Take 0.3 mL of 10% H2SO4 and 0.75 mL of 10% Triton X-100. Add H2O to 10 mL. The solution will be stable for 1 wk at room temperature.

2. AB reagent (Alcian blue in 0.1% H2S04/0.25% Triton): Take 5.0 mL of Alcian blue stock solution and add 1.0 mL of 10% H2S04 and 2.5 mL of 10% Triton. Add H20 to a final volume of 100 mL. The solution will be stable for 1 wk at 4°C.

3. DMSO washing solution (40% (v/v) DMS0/0.05M MgCl2): Add 40 mL of DMSO to 40 mL of H20 and add 1.7 mL of 3.0 M MgCl2. Add H20 to 100 mL. Store at room temperature. The solution will be stable for 1 mo.

4. Ethanol washing solution (50% (v/v) ethanol/0.05M MgCl2): Take 52 mL of ethanol (96%, v/v) and add 1.7 mL of 3.0 MMgCl2. Add H2O to 100 mL. Store at room temperature. The solution will be stable for 1 mo.

5. Gu-prop-H2O: 4 M guanidine-HCl/33% 1-propanol/0.25% Triton/0.1% Ficoll.) Take 50 mL of 8.0 M guanidine-HCl, 33 mL of 1-propanol p.a., and 2.5 mL of 10% Triton. Add 0.1 g of Ficoll. Mix and add H2O to 100 mL. Do not use 2-propanol (isopropanol)! Store at room temperature. The solution will be stable for 1 yr.

6. Prop-gu: 71% 1-propanol/2.3 M guanidine-HCl): Mix 71 mL of 1-propanol p.a. and 29 mL of 8.0 M guanidine-HCl. Do not use 2-propanol (isopropanol)! Store at room temperature. The solution will be stable for 1 yr.

Table 1

Preparation of Calibrators, Tube Absorbance Assay

Calibrator (mg/mL)

Dilution

0.05

0.025

80^1 CsC stock 1000^1 0.4mg/mL 1000^1 0.2mg/mL 1000^1 0.1mg/mL 1000^1 0.05mg/mL 1000^1 0.025mg/mL

1920 1000^1 1000^1 1000^1 1000^1 1000^1

0.0125

7. Prop-Tris: 75% 1-propanol/0.1 MTris-Ac. Mix 75 mL of 1-propanol and 10 mL of 1.0 M Tris/Ac, pH 7.4. Add H20 to 100 mL. Store at room termperature. The solution will be stable for 1 yr.

2.3. Sample Preparation

GAGs are stable at 4°C, in the absence of cells. The protein cores of proteoglycans may be degraded by proteolytic enzymes. The sample must not contain any particles that sediment during centrifugation or that are insoluble in 0.4 M guanidine-HCl at pH 1.5. Cell debris and insoluble material should be removed by centrifugation (12,000g for 15 min). If the sample does contain material that precipitates in 0.4 M guanidine-HCl at pH 1.5, it must be removed by a preceding step (see Subheading 3.4.).

Samples such as plasma or synovial fluid with a high protein and a low proteoglycan content may be concentrated and purified with an extra preceding Alcian blue precipitation. (see Subheading 3.5.)

3. Methods

3.1. Quantitation, Tube Absorbance Assay (Table 1)

The GAG/PG are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and collected by centrifugation. Excess stain in the pellet is removed by washing in DMSO. The GAG/PG-Alcian blue complexes are dissolved and dissociated in a 4 M guani-dine-HCl/propanol mixture. The amount of GAG/PG is directly proportional to the Alcian blue concentration measured as absorbance at 605 or 620 nm. The tube/absor-bance assay has a measuring range of 1-20 ^g of GAG. Sample volumes given are only suggestive. The method can be applied at any scale as long as the final concentration of guanidine-HCl is 0.4 mol/l at pH 1.5 - 2.0.

1. Put 50 ^L of either blank (water), sample, calibrator, or control in 2-mL polypropylene centrifuge tubes.

2. Add 50 ^L of 8 MGu-HCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl are analyzed without further addition of guanidine-HCl. Take 100 ^L of the sample and go directly to step 3.

3. Add 50 ^L of SAT reagent. Mix and leave for 15 min.

4. Add 750 ^L of cold AB reagent. Mix and leave for 15 min to overnight at 4°C.

5. Centrifuge for 15min at 12,000g. Remove supernatant and discard. Care should be taken that pellets are left intact when supernatants are removed by suction. The use of a manu-

mg/ml

Fig 1. Tube assay of different GAG in blood plasma. Commercial preparations of glycosami-noglycans were dissolved in water or in blood plasma (0-0.4mg/mL) and was quantitated with Alcian Blue using the tube assay. -□- Chondroitin-6-sulphate; -O- Keratan sulphate; -O-Heparan sulphate; Open symbols: GAG dissolved in water. Closed symbols: GAG dissolved in blood plasma.

ally operated syringe for suction and immediate removal of the supernatants after cen-trifugation is therefore recommended. The tube should be held at an angle and in such a way that the pellet and needle are visible during the entire operation. The needle should be gradually lowered with the meniscus during suction and never be allowed to touch the pellet.

6. Add 500 ^L of DMSO solution to the pellet. Mix thoroughly until the pellet is suspended in the washing solution. Mix for 15 min on a shaker.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 500 ^L Gu-prop-H2O solution to the pellet. Mix for 15 min on a shaker. Check that the pellet is completely dissolved.

9. Dispense 2 x 240 ^L of the supernatant into an ELISA-microplate. Read absorbance at 605 nm.

10. Plot the absorbances against amount of GAG in each calibrator (0-20 ^g). The plot should be a perfectly straight line with an absorbance of approximately 2.0 at 20 ^g and blank values below 0.05 (Fig. 1). High blank values may be caused by insoluble, degraded stain. The dry Alcian blue stain has a limited shelf life, and some batches are not suitable for biochemical work. The stock solution also has a limited shelf life, and it is important that the AB stock solution be centrifuged in order to remove the insoluble portion of Alcian blue. Fit a linear equation and calculate the amount of GAG in each sample. Alternatively, if only a single calibrator concentration is used, a factor is calculated by dividing the calibrator (^g) by the corresponding absorbance. The absorbance of the unknown samples is then multiplied by the factor to obtain micrograms of GAG in each sample.

All commercial sulfated GAG samples (CsA, CsC, Ds, Ks , Hs) should give a similar colour yield. HA, Hyaluronan, DNA, and RNA do not react with Alcian blue at all. There is no interference from proteins in blood plasma (Fig. 1).

3.2. Preparation for Electrophoresis Without Removal of Alcian Blue

The GAG/PGs are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and collected by centrifugation. Excess stain in the pellet is removed by washing in DMSO.

Fig. 2. Alcian blue precipitation without stain removal. Proteoglycans in 4 M Gu-HCL extracts of cartilage and skin were precipitated with by Alcian blue as described under Subheading 3.2. The samples were analyzed by electrophoresis in 1% agarose and stained with AB reagent containing 0.4 M GuHCl. Lane 1; chondroitin-6-sulfate. Lane 2; 4 M Gu-HCl extract of bovine nasal cartilage; Lane 3; 4 M Gu-HCl extract of human skin. The position of AB-SDS complexes is indicated in the figure.

Fig. 2. Alcian blue precipitation without stain removal. Proteoglycans in 4 M Gu-HCL extracts of cartilage and skin were precipitated with by Alcian blue as described under Subheading 3.2. The samples were analyzed by electrophoresis in 1% agarose and stained with AB reagent containing 0.4 M GuHCl. Lane 1; chondroitin-6-sulfate. Lane 2; 4 M Gu-HCl extract of bovine nasal cartilage; Lane 3; 4 M Gu-HCl extract of human skin. The position of AB-SDS complexes is indicated in the figure.

The GAG/PG/Alcian blue complexes are dissolved directly in an SDS buffer and analyzed by electrophoresis.

The SDS used to dissolve the GAG/PG-Alcian blue complexes will move in front of any GAG/PG, Fig. 2. The relative mobility of the SDS-Alcian blue complexes will depend on the ratio of SDS bound per Alcian blue molecule.

This method is useful for revealing losses during the propanol precipitation step (see step 9 and 10 under Subheading 3.3.). Any losses can be detected by comparing the electrophoresis patterns of the same samples obtained as described under Subheading 3.2. and 3.3.

1. Take 100 ^L of either blank (water), sample, calibrator or control to 2 mL polypropylene centrifuge tubes.

2. Add 100 ^L of 8M GuHCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl are analyzed without further addition of guanidine-HCl. Take 200 ^L of the sample and go directly to step 3.

3. Add 100 ^L of SAT reagent. Mix and leave for 15 min.

4. Add 1500 ^L of cold AB reagent. Mix and leave for 15min - overnight at 4°C.

5. Centrifuge for 15min at 12000g. Remove supernatant and discard.

6. Add 750 ^L of DMSO solution to the pellet. Mix thoroughly; the pellets should be suspended in the washing solution. Mix for 15 min.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 50 ^L of TG x 3/2.5% SDS to pellets (sample, calibrator and control).

9. Quantitation may be performed as follows. Put 5 ^L of each dissolved pellet on a microtiter plate. Add 100 ^L TG x 3/0.5% SDS to each well and mix. Use reverse pipetting technique to minimize formation of bubbles. Read absorbance at 600 nm.

10. The amount of sample to be applied to each lane is calculated as follows. Divide the calibrator (^g) by the corresponding absorbance. The absorbance of the unknown sample is then multiplied by the factor to obtain micrograms of GAG in each sample. About 3-10 ^g are suitable for electrophoresis. Add TG x 3/0.5% SDS to a final volume of 50 ^L.

11. Add 25 ^L of liquified 1% agarose with 15% glycerol. Heat at 105°C for 5 min and apply 75 ^L of the hot sample on each lane on a 1% agarose gel in 0.1 M Tris-Ac. Electro-phorese for 1 h at 100 V, until the BFB front has moved 60 mm. Stain in AB-reagent with 0.4 M GuHCl (see Fig. 2.).

3.3. Preparation with Removal of Alcian Blue

The GAG/PGs are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and collected by centrifugation. Excess stain in the pellet is removed by washing in DMSO. The GAG/PG-Alcian Blue complexes are dissociated and dissolved in a 4 M guanidine-HCl/ propanol mixture. The GAG/PGs are precipitated by increasing the propanol concentration, with addition of a 71% propanol/guanidine-HCl mixture, and are then recovered by centrifugation. The amount of GAG/PG is directly proportional to the Alcian blue concentration in the supernatant, measured as absorbance at 605 nm.

1. Put 100 ^L of either blank (water), sample, calibrator, or control in 2-mL polypropylene centrifuge tubes.

2. Add 100 ^L of 8 M Gu-HCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl are analyzed without further addition of guanidine-HCl. Take 200 ^L of the sample and go directly to step 3.

3. Add 100 ^L of SAT reagent. Mix and leave for 15 min.

4. Add 1500 ^L of cold AB reagent. Mix and leave for 15min to overnight at 4°C.

5. Centrifuge for 15 min at 12000g. Remove supernatant and discard.

6. Add 750 ^L of DMSO solution to the pellet. Mix thoroughly; the pellets should be suspended in the washing solution. Mix for 15 min.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 250 ^L of Gu-prop-H2O to the pellet. Mix for 15 min or until the pellet is dissolved.

9. Add 1750 ^L of Prop-gu. Mix and leave for 60 min . Loss of GAG/PG may occur during propanol precipitation/centrifugation. Most proteoglycans will precipitate quantitatively at a rather low propanol concentration but isolated GAG chains may not precipitate quantitatively. Thus, keratan sulfate require higher propanol concentrations than other GAGs, Fig. 3. Any losses can be detected by comparing the electrophoresis patterns of the same samples without propanol precipitation (see Subheading 3.2.) and with propanol precipitation (see Subheading 3.3.).

10. Centrifuge for 15 min at 12000g. Remove the supernatant using a syringe. Read absor-bance at 605 nm of the supernatant. The expected absorbance of the supernatant is 1.0 at 0.4 mg/mL (40 ^g).

11. Add 750 ^L of Prop-Tris to the pellet. Mix.

12. Centrifuge for 5 min at 12,000g. Remove the supernatant and discard. The pellet will always contain some insoluble degraded Alcian blue. The dye in the pellet is not water soluble, and can be sedimented by centrifugation. The concentration of propanol in the Prop-gu solution, used for precipitation of GAG/PG, will affect the amount of Alcian blue remaining in the pellet, Fig. 4. The amount of Alcian blue in the pellet also depends on the GAG and is especially high with keratan sulfate, Fig. 4.

13. Dissolve the pellet in 50 ^L TG x 3/0.1% SDS. The pellet may also be dissolved in water or a buffer without SDS.

14. The amount of proteoglycan needed for electrophoresis may be calculated as above (see Subheading 3.2., step10) and analyzed by electrophoresis (see Fig. 5).

20.0 10.0

Fig. 3. Removal of Alcian blue stain: precipitation of GAG at different concentrations of propanol. GAGs were precipitated by Alcian blue and the precipitates were dissolved in 250 ^L of a 4 M GuHCl/33% propanol mixture as described under Subheading 3.3. GAGs were precipitated by adding 1750 ^L of a propanol/GuHCl mixture. The following propanol/GuHCl mixtures were used: 67% propanol 2.66 M GuHCl; 69% propanol/2.48 M GuHCl; 71% pro-panol/2.32 M GuHCl; 73% propanol/2.16 M GuHCl; 75% propanol/2.0 M GuHCl. The precipitated GAGs, recovered by centrifugation, were again quantitated using the method of Subsection 3.1. and the recovery calculated. -□- Chondroitin-6-sulfate; -O- Keratan sulfate; -O- Heparan sulfate.

Fig. 3. Removal of Alcian blue stain: precipitation of GAG at different concentrations of propanol. GAGs were precipitated by Alcian blue and the precipitates were dissolved in 250 ^L of a 4 M GuHCl/33% propanol mixture as described under Subheading 3.3. GAGs were precipitated by adding 1750 ^L of a propanol/GuHCl mixture. The following propanol/GuHCl mixtures were used: 67% propanol 2.66 M GuHCl; 69% propanol/2.48 M GuHCl; 71% pro-panol/2.32 M GuHCl; 73% propanol/2.16 M GuHCl; 75% propanol/2.0 M GuHCl. The precipitated GAGs, recovered by centrifugation, were again quantitated using the method of Subsection 3.1. and the recovery calculated. -□- Chondroitin-6-sulfate; -O- Keratan sulfate; -O- Heparan sulfate.

66 67 68 69 70 71 72 73 74 75 76

Fig. 4. Removal of Alcian blue stain: coprecipitation of Alcian blue with GAG at different concentrations of propanol. Same experiment as in Fig. 3. The amount of Alcian blue that co-precipitated with GAGs after centrifugation was quantitated as absorbance at 620 nm of the solubilized pellet. -□- Chondroitin-6-sulfate; -O- Keratan sulfate; -O- Heparan sulfate.

Fig. 5. Alcian blue precipitation with stain removal. Proteoglycans in four different human synovial fluids were precipitated by Alcian blue as described under Subheading 3.3. The samples were analyzed by electrophoresis in 1% agarose and stained with AB-reagent containing 0.4 M Gu-HCl.

3.4. Purification of Difficult Samples

Some samples may contain material that precipitates at a low pH or that binds unspecifically to Alcian blue. Such material may be removed by an extra Alcian blue precipitation step as described below.

1. Put 100 ^L of either blank (water), sample, calibrator, or control into 2-mL polypropylene centrifuge tubes.

2. Add 100 ^L of 8 M Gu-HCl. Mix and leave for 15 min.

3. Add 100 ^L of SAT reagent. Mix and leave for 15 min.

4. Add 1500 ^L of cold AB reagent. Mix and leave for 15 min to overnight at 4°C.

5. Centrifuge for 15 min at 12000g. Remove supernatant and discard.

6. Dissolve the pellet in 100 ^L of H2O + 100 ^L of 8 M GuHCl + 100 ^L of SAT reagent. Mix for 15 min.

7. Centrifuge for 15 min at 12000g. Transfer the supernatant to a new vial. Discard the pellet. Alternatively, the pellet may be washed in DMSO-washing solution and dissolved in TG x 3/2.5% SDS and analyzed by electrophoresis to verify that no GAG/PG are present in the pellet, Fig. 6.

8. Continue at step 4 under Subheading 3.2. or 3.3.

3.5. Large-Scale Preparation

Large-scale preparation consists of two successive precipitations with Alcian blue and is suitable for dilute samples such as plasma and urine. In the first precipitation step the GAG/PGs are concentrated. When the first precipitate is dissolved and centrifuged, any acid-insoluble material is left behind in the pellet. The supernatant will contain the proteoglycans, which are further purified by a second Alcian blue precipitation.

The GAG/PG-Alcian blue complexes recovered after the second precipitation are dissolved and dissociated in a 4 M guanidine-HCl/propanol mixture. The GAG/PG is recovered free from stain by selective propanol precipitation.

Table 2

Dispensed

Tota1

Tota1

Approx.

vo1ume

vo1ume

absorbance

amount of

Fraction

Absorbance

(mL)

(mL)

GAG/PG ^g

Step 5—supernatant

1.86

0.1

3.75

69.7

348

Step 8—supernatant

0.51

0.2

10

25.5

127

Step 11—supernatant

1.97

0.24

6.8

55.8

279

Fina1 quantitation of 100 ^L1.75 x 5

0.24

0.5

45.5

227

Fig 6. Purification of difficult samples. Proteoglycans in human blood plasma were precipitated with Alcian blue and the pellets extracted with a mixture of 4 M Gu-HCl/SAT-reagent and centrifuged as described under Subheading 3.4. The extracted proteoglycans in the supernatant were reprecipitated as described under Subheading 3.2. The pellet was washed in DMSO washing solution and dissolved in TG x 3-2.5% SDS. The samples were analyzed by electrophoresis in 1% agarose and stained with AB-reagent containing 0.4 M GuHCl. Lane 1; supernatant from Subheading 3.4., step 7; Lane 2; pellet from Subheading 3.4., step 7. The position of AB - SDS complexes is indicated in the figure.

The amount of GAG is directly proportional to the Alcian blue concentration in the supernatant, measured as absorbance at 605 or 620 nm.

Recovery of GAG/PG throughout the procedure is approximately 70%. The main losses are in the propanol precipitation/centrifugation step (see Table 2). The size distribution and purity of the purified preteoglycans could be analyzed by agarose electrophoresis and parallel lanes stained with Coomassie Brilliant blue and Alcian blue, Fig. 7. The proportions given below can be applied at any scale.

1. Put a 25 mL sample in a 50 mL propylene tube. Add 2.5 mL 8 M Gu-HCl and 1.25 mL 10% Triton. Mix and leave for 15 min.

2. Add 2.5 mL of the Alcian blue stock solution. Mix carefully and leave for 15 min.

3. Add 0.67% (v/v) H2SO4 up to 50 mL. Leave overnight at 4°C.

4. Centrifuge at 3300g, 60 min. Remove the supernatant and discard. Let the tube drain upside down.

Fig. 7. Large scale preparation. Proteoglycans were prepared from human blood platelets with two successive precipitations with Alcian Blue as described under Subheading 3.5. The purified proteoglycans were analyzed by electrophoresis in 1% agarose.Lane A: stained with AB - reagent with 0,4 M GuHCl. Lane B: stained with Coomassie Brilliant Blue in 5% (v/v) acetic acid - 25% (v/v) ethanol.

Dot Blot Apparatus

Fig. 8. Dot-blot assay. Heparan sulfate calibrator (0-80 mg/L), dissolved in water (A) or in blood plasma (B), was precipitated with Alcian blue and the GAG precipitates collected on a PVDF membrane by filtration in a dot-blot apparatus. The membrane was scanned on a single layer of clear plastic.

5. Add 1.25 mL of 8 MGu-HCl, and 1.25 mL of H2O, and 1.25 mL of SAT reagent. Mix for 15 min. Centrifuge at 3300g for 15min. Transfer the supernatant to another tube. Determine absorbance of a suitable volume at 605 nm.

6. Add 22.5 mL of AB reagent. Leave for 2 h. Centrifuge at 3300g for 1h. Remove the supernatant and discard. Let the tube drain upside down.

7. Add 10 mL of DMSO washing solution and mix until the pellet is suspended. Mix for 15 min.

8. Centrifuge at 3300g for 20 min. Remove the supernatant and determine absorbance of a suitable volume at 605 nm. Let the tube drain upside down.

9. Dissolve the pellet in 1000 ^L of Gu-prop-H2O solution (prepared without Ficoll). Mix for 30 min or until the pellet is completely dissolved. Divide into four 2 mL propylene vials (approximately 250 ^L/vial).

10. Add 1750 ^L of Prop-gu solution. Leave for 1h to precipitate the GAG/PG.

11. Centrifuge at 12,000g for 15 min. Remove the supernatants carefully, using a syringe, to new tubes. Determine absorbance of a suitable volume at 605 nm. Loss of GAG/PG may occur during the propanol precipitation/centrifugation in steps 10-11. Such losses are revealed by comparison of the total absorbances calculated in step 11 and 13 (see Table 2).

12. Add 750 ^L of Prop-Tris solution to the pellet. Mix. Centrifuge at 12,000g for 15 min. Remove the supernatant and discard. Let the tube drain upside down. Enzymatic degradation of GAG/PG is inhibited if propanol is present in the pellet. Propanol may be evaporated by drying at 37°C. Too intensive drying should be avoided, since the GAG/PG may be difficult to redissolve.

13. Dissolve the pellet in a suitable buffer (see Note 1). Take a suitable volume for quantitation (see Subheading 3.1.). Calculate total absorbance and the amount of GAG/PG.

Calculation. The recovery of GAG/PG in each step may be monitored by taking the absorbance of a suitable portion and calculating the total absorbance. Care should be taken not to exceed the actual absorbance range of the instrument since most ELISA photometers are only reliable below an absorbance of 2.0. Calculate the total absorbance as follows: total volume of supernatant /volume in ELISA well x A605. The approximate amount of GAG/PG is obtained by multiplying by 5.

The following results in Table 2 were obtained when 250 ^g of shark CsC were dissolved in 25 mL of human blood plasma. The absorbance was measured in each of the steps indicated above. The purified GAG/PG was dissolved in 500 ^L 4 M guanidine-HCl and 100 ^L were taken for final quantitation (see Subheading 3.1.).

Comments: The figure obtained for step 5—supernatant overestimates the amount of GAG/PG since it also contains some co-precipitating dye. This dye is removed by washing in DMSO. The figure obtained for step 8—supernatant overestimates the amount of dye washed away since the molar absorbance coefficient of Alcian blue is higher in DMSO than in guanidine-HCL solution. The figure obtained for step 11— supernatant is always higher than that obtained by final quantitation of the redissolved purified GAG/PG sample. This is presumably because of GAG/PG losses during the propanol precipitation/centrifugation in step 11 or by incomplete solubilization in step 13.

3.6. Dot-Blot Reflectance Assay (see Table 3)

The Alcian blue-GAG complexes are collected on a PVDF membrane, by filtration in a dot-blot apparatus, and the stain quantitated as reflectance by scanning and densi-tometry. The assay requires 10 ^L of sample and has a measuring range of 10-800 ng

Table 3

Dot-Blot Reflectance Assay

Table 3

Calibrator

Dilution

H2O

GAG in 10^l

80

16 ^L CsC stock

1984

800

40

1000 ^L 80 mg/L

1000

400

20

1000 ^L 40 mg/L

1000

200

10

1000 ^L 20 mg/L

1000

100

5

1000 ^L 10 mg/L

1000

50

2.5

1000 ^L 5 mg/L

1000

25

1.25

1000 ^L 2.5 mg/L

1000

12.5

of GAG. No interference from plasma proteins is evident when chondroitin sulfate, keratan sulfate or heparan sulfate is dissolved in a blood plasma with low endogenous GAG content (see Fig. 8). The dot-blot assay is sensitive enough to measure the amount of GAG released upon coagulation; that is serum values (see Fig. 10) are much higher than plasma values (see Fig. 9).

1. Put 10 ^L of either blank (water), sample, calibrator, or control in duplicate in a 96-well polystyrene microplate. All six calibrator levels, plus blank, should be used, as the calibration curve is nonlinear.

2. Add 20 ^L of a 1/1 mixture of 8 M GuHCl and SAT reagent.

3. Mix on a microplate shaker at 200 rpm for 15 min.

4. Add 200 ^L of cold AB reagent. Mix as above for 60 min.

5. Assemble a 96-well MilliBlotD apparatus according to the instructions of the manufacturer, using a PVDF membrane wetted and blocked for 1 h in 1% (v/v) Triton X-100.

6. Add 200 ^L of 0.4 M Gu-HCl/0.1% (v/v) sulfuric acid/0.25% (v/v) Triton X-100 to each well immediately upon assembly (see Note 2). Evacuate approximately 100 ^L by suction. Close the outlet tubing with a rubber stopper.

7. Transfer 200 ^L of the samples from the microplate using an eight-channel pipet and the reverse pipetting technique. Precipitates of GAG/PG-Alcian blue may form during incubation in step 4. Any precipitates present at this point can be dispersed by repetitive pipetting in the microplate well before transfer.

8. Evacuate the wells by suction.

9. Add 200 ^L of 50% (v/v) ethanol in 0.05 M MgCl2 and evacuate the wells by suction. Repeat once

10. Remove the membrane from the apparatus and wash briefly in distilled water before air drying. High blank values are caused by degraded Alcian blue stain. Degradation may occur both in dry form and in liquid preparations (stock or AB reagent).

11. Scanning. Scanning is performed in the reflectance color mode with the gamma curve set at 1.0 and the dynamic range adjusted by positioning the 256 gray scales over the actual range displayed in the histogram window. The membrane is placed on top of one transparent plastic sheet in order to increase the sensitivity. The green and blue channels are discarded. The red channel is saved in gray-scale mode, Fig. 8.

12. Densitometry. The Scan Analysis software for Macintosh from Biosoft® (Cambridge, UK) is used for densitometry (see Note 3). The manual mode is used without background subtraction, and the densitometry is performed on a rectangle encompassing two dots of a

Chondroitin Sulphat Assay

Fig. 9. Dot-blot assay of different GAGs in serum. Commercial preparations of glycosami-noglycans were dissolved in water or in serum (0-80 mg/L) and quantitated with Alcian blue using the dot-blot assay-O- Chondroitin-6-sulfate; ♦ Keratan sulfate; -O- Heparan sulfate; open symbols, GAG dissolved in water. Closed symbols, GAG dissolved in serum.

Fig. 9. Dot-blot assay of different GAGs in serum. Commercial preparations of glycosami-noglycans were dissolved in water or in serum (0-80 mg/L) and quantitated with Alcian blue using the dot-blot assay-O- Chondroitin-6-sulfate; ♦ Keratan sulfate; -O- Heparan sulfate; open symbols, GAG dissolved in water. Closed symbols, GAG dissolved in serum.

0 10 20 30 40 50 60 70 80 90

mg/ml

Fig. 10. Dot-blot assay of different GAGs in blood plasma. Commercial preparations of gly-cosaminoglycans were dissolved in water or in blood plasma (0-80 mg/L) and quantitated with Alcian blue using the dot blot assay. -O- Chondroitin-6-sulphate; ♦ Keratan sulphate; -O-Heparan sulphate; open symbols, GAG dissolved in water; closed symbols, GAG dissolved in blood plasma.

duplicate sample. The non linear relationship between reflectance and concentration is evident (see Figs. 9 and 10).

13. Curve fitting. The arbitrary units obtained in Scan Analysis are pasted into the Ultra Fit software from Biosoft® and multiplied by 0.01 in order to get manageably sized factors (see Note 3). The densitometry units (x axis) are plotted against the calibrator values (y axis). The Cubic equation (a third-degree polynomial) is used for curve fitting. In order to prevent minima appearing within the fitted curve, parameter c should be restricted to values > 0. The fitted equation is used to calculate the concentration of unknown samples.

4. Notes

1. The pellet will always contain some degraded Alcian blue, which is not water soluble and can be sedimented by centrifugation.

2. Uneven spots caused by air bubbles present underneath the membrane are prevented by always having excess fluid beneath and above the membrane when assembling the apparatus and applying the samples. Therefore, buffer should be added to each well immediately upon assembly.

3. Densitometry and curve fitting may be performed with any suitable software. A third-degree polynomial or any other suitable equation may be used for curve fitting.

References

1. Scott. J. E. (1970) Histochemistry of Alcian blue I. Metachromasia of Alcian Blue, Astrablau and other cationics and other phthalocyanin dyes. Histochemie 21, 277-285

2. Bjornsson, S. (1993) Simultaneous preparation and quantitation of proteoglycans by precipitation with Alcian Blue. Anal. Biochem. 210, 282-291.

3. Bjornsson, S. (1993) Size dependent separation of proteoglycans by electrophoresis in gels of pure agarose. Anal. Biochem. 210, 292-298.

4. Bjornsson, S. (1998) Quantitation of proteoglycans as glycosaminoglycans in biological fluids using an Alcian Blue dot blot analysis. Anal. Biochem. 256, 229-237.

5. Farndale, R. W., Sayers, C. A., and Barrett A. J. (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res. 9, 247-248.

6. Farndale, R. W., Buttle, D. J., and Barrett A. J. (1986) Improved quantitaion and discrimination of sulphated glycosaminoglycans by use of dimethylmetylene blue. Biochim. Biophys. Acta. 883, 173-177.

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