Approximately 30 mg (wet weight) biopsies of normal tissue and carcinoma-afflicted tissue that also contained surrounding stroma were freeze dried and digested for 48 h at 55 °C in 50 mM Tris/HCl, pH 8, 1 mM CaCl2, 1% Triton X-100, containing 0.5 mg pronase. Glycosaminoglycans (GAGs) were released by incubation of the pronase-digested tissue sample with 0.5 M NaOH at 4 °C for 24 h. The alkaline solution was neutralized to pH 6 by acetic acid and centrifuged at 11,000 × g for 10 min. The supernatants containing GAGs were recovered by a weak anion exchange workup, using 1.5 ml DEAE-Sephacel columns. Samples were loaded, and washed with 25 mL 0.1 M NaCl, 20 mM NaAc pH 6.0 buffer. GAGs were eluted with 2.5 mL of 1 M NaCl, 20 mM NaOAc pH 6.0. These fractions were desalted with PD-10 columns and vacuum dried. The exhaustive depolymerization was accomplished by dissolving one sixth of each sample in 10 μl water, followed by adding 8 μl 100 mM Tris/HCl pH 7.45 2 μl 1 M ammonium acetate, 20 mU of chondroitinase
