In electrophoretic separations, proteins are solubilized in buffers containing SDS, an anionic detergent. Thus macromolecular complexes containing monomeric species that are loosely bound either by electrostatic (including hydrogen bonding) or hydrophobic interactions are disrupted, leading to complex degradation on gels. However, such macromolecular complexes can be separated in an intact configuration by size-exclusion gel filtration chromatography.
For gel filtration chromatography, pooled tissue samples were macerated using a pestle and mortar in Tris-HCl buffer (100 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.02% sodium azide, pH 7.5). The extract was centrifuged at 10,000g for 5 minutes, and the supernatant was removed for analysis. Altogether four preparations (preparations 1–4) were obtained (preparation 1, from two eyes, donor ages 72 and 79 years; preparation 2, from three eyes, donor ages 71, 73, and 84 years; preparation 3, from two eyes, donor ages 67 and 81 years; and preparation 4, from eight eyes, donor ages 69–84 years). Extracted supernatant volumes ranged from 1.5 mL for preparation 1 to 3 mL for preparation 4.
Supernatant aliquots were applied to a filtration column (30 cm × 1.5 cm inner diameter; Sepharose CL-6B; Sigma-Aldrich) pre-equilibrated with Tris-HCl buffer. Flow rate was adjusted to 0.6 mL/ min, and fractions were collected every 2 minutes for 1.5 to 2 hours. The protein profile of the chromatography run was obtained by measuring the absorbance of the fractions at a wavelength of 280 nm. Fraction aliquots were then processed for zymographic analysis as described earlier. The intensity of the bands of individual gelatinases was plotted as a function of fraction number to deter mine their mobilities on the column.