Improved and simple microassay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies (1)
Most sulfated glycosaminoglycans (GAGs) chains are covalently linked to core proteins to form proteoglycans, which are strategically located at the cell surface and in the extracellular matrix. Proteoglycans and GAGs play different roles in diverse processes, such as enzyme regulation and cellular adhesion, growth, migration, or differentiation.
The conditions that increased complexation of dye to sulfated GAGs by using a DMMB solution prepared in a formate buffer pH 3.0 containing 5% ethanol and 0.2 M guanidine hydrochloride (GuHCl). The use of ethanol at low pH allowed exhaustive complex precipitation within 30 min when vigorous shaking was applied during this time. The GAG–DMMB complex was obtained as a stable pellet after centrifugation of the treated sample, and its isolation was particularly easy by simple decantation of soluble materials.
Using a decomplexation 4 M GuHCl solution at pH 6.8 containing 10% propan-1-ol. Dissociation was favored at neutral pH because the cationic state of the dye, which induced complexation to polyanionic GAGs, was highly reduced at this pH. The high GuHCl concentration allowed GAG solubility favoring its dissociation from the complex. 10% porpan-1-ol in the decomplexation solution to enhance DMMB signal at 656 nm was included. The DMMB complexation/decomplexation protocol showed two peaks of absorption, at about 610 nm and 656 nm, in samples.
With consisting of the preparation of a 32 mg/L solution followed by twofold dilution in the ethanol/GuHCl formate buffer, the DMMB solution obtained stable for more than 4 months when stored at room temperature in the darkness. Like sulfated GAGs, DNA readily forms complexes with the dye, absorbing at nearly the same wavelength as the GAG–dye complex. Some authors suggested the use of GuHCl (up to 0.24 M) in the complexation solution to decrease these DNA/dye interactions. Low GuHCl concentration (0.08 M) did not avoid interaction of DNA with DMMB and thus disturbed GAG determination. Another way to overcome DNA–dye interaction was to eliminate or highly reduce DNA by filtration of samples after proteinase K treatment. GuHCl concentration may be increased to 0.20 M without affecting GAG–DMMB complexation.
The linear regression curves obtained by plotting the absorbance values of DMMB in solution after decomplexation from CS versus its nominal ng or µg/ml concentration. For submicrogram quantities of GAGs (0.025–1.1 µg/ml), the calibration curve (a) was used. For samples containing microgram quantities of GAG (0.5–5 µg/ml), the calibration curve (b) was applied. Linearity at both low and high quantities of GAGs was excellent and was demonstrated up to 10 µg/ml.
Preparation of DMMB solution
Preparation of the DMMB solution was derived from that reported by Farndale et al. (1982) with some modifications. In brief, 16 mg DMMB were dissolved in 25 ml ethanol and filtered through filter paper. One hundred milliliters of 1 M GuHCl, 1g sodium formate, and 1 ml 98% formic acid were then added to the DMMB ethanolic solution, and the final volume was completed to 500 ml with distilled water. This solution was unstable and needed to be rapidly diluted (1:1) with the formate solution prepared as described but without DMMB. This DMMB solution was stable for at least up to 4 months when stored at room temperature in the darkness.
DMMB decomplexation solution
A 50 mM sodium acetate solution buffer (pH 6.8) containing 10% propan-1-ol was prepared and used to solubilize powdered GuHCl to a final concentration of 4 M. This solution was stable for at least 4 months at room temperature.
Spectroscopic determination of sulfated GAG: GAG–DMMB complexation/decomplexation
The content of sulfated GAGs was determined using the DMMB solution as follows. 1 ml working DMMB solution was added to 100 µl proteinase K–treated sample, and the mixture was vigorously vortexed for 30 min to promote complete complexation of the GAG with DMMB. The insoluble GAG–DMMB complex was then separated from the soluble materials, including DMMB excess, by centrifugation (12,000 × g, 10 min). The supernatant was discarded, and the pellet was dissolved with the decomplexation solution. The added volume of this solution was adjusted according to the quantity of GAGs. For samples containing GAGs at the microgram level, 1 ml decomplexation solution was added. For lower quantities (<1.0 µg) 500 µl was added. Decomplexation was achieved by shaking the mixture for 30 min. Absorbance was measured at 656 nm.
Quantification of HS
HS was eliminated from the original sample. 100 µl proteinase K–treated sample was mixed with 100 µl sodium nitrite (5%) and 100 µl acetic acid (33%). Samples were gently shaken and kept at room temperature for 1 h. To stop the reaction, 100 µl ammonium sulfamate (12.5%) was added, and the mixture was shaken for a further 5 min. Remaining sulfated GAGs were determined in 100 µl of this nitrous acid reaction mixture by following the DMMB protocol as described. The GAG remaining in the sample represented O-sulfated GAGs, including CS. The N-sulfated GAGs (HS) content was then calculated as the difference between the total GAGs and the O-sulfated GAGs in each sample.