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The noncollagenous domain of basement membrane collagen exists as a hexamer upon excision with bacterial collagenase. Two hexamer subtypes, differing in subunit composition, have been identified and several additional subtypes are possible because at least two, and possibly more, triple-helical molecules, differing in chain composition, exist in authentic basement membranes (Saus J, Wieslander J, Langeveld JPM, Quinones S, and Hudson BG. (1988) Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV. J. Biol. Chem. 263:13374-13380). In the present study, the physiochemical behavior of hexamer during two-dimensional electrophoresis was evaluated. The hexamers from three different membranes of bovine origin (lens capsule, glomerular, and placenta) were found to exhibit an unusual dissociative property during the pH gradient electrophoresis used in the first dimension; namely, the hexamers dissociate under nondenaturing conditions into monomer and dimer subunits concomitant with the resolution of subunits. This dissociative property provided the basis for a new procedure using chromatofocusing for the preparative resolution of hexamer subunits with retention of their native structure and capacity to associate into a hexamer configuration. Associative studies revealed that the capacity for hexamer assembly is contained within the monomer subunit, a property which may be of fundamental importance in the mechanism of the assembly of collagen IV protomers and the association of protomers forming a supramolecular structure.