Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue, an unmanageable starting material for conventional column chromatography (1)
A filter paper-based (FPB) DEAE-cellulose column chromatog- raphy can simplify the protein purification from the start material which requires tedious preliminary fractionation before the chromatographic procedure. The type V collagen is a member of fibril-forming collagens (Types I, II, III, V, and XI) and consists of three subunit chains designated a1(V), a2(V), and a3(V). At least two submolecular species, [a1(V)]2a2(V) and a1(V)a2(V)a3(V) were identified.
Type I collagen was recovered in the non-adsorbed fraction (peak A) and type V collagen and pepsin were eluted with 0.3 M (peak B) and 1.0 M NaCl (peak C), respectively. The a1(V), a2(V) subunits and their oligomers are detected by SDS–PAGE analysis. On the other hand, no significant amount of the a3(V) subunit was detected.
The type V collagen-rich fraction obtained by the FPB DEAE-cellulose column was dialyzed against the equilibrium buffer of the Baker- bond WP-CSX column and applied to the small Bakerbond WP-CSX column. Type I and V collagen were eluted with 170 mM (peak B) and 500 mM NaCl (peak C), respectively.
Type I collagen was eluted in the non-adsorbed fraction (fraction A) and type V collagen was eluted by the gradient of NaCl (fractions B and C). The type V collagen in the effluent could be recovered as a precipitate by dialysis against water or mixing with cold ethanol to give 35% (v/v). Two subunit chains with electrophoretic mobility corresponding to the a1(V) and a2(V) chains were resolved by the anion-ex- change HPLC.
A small peak (peak B) was detected after the elution of a1(V) and a2(V) (peak A), which corresponds to the elution position of a1(XI). How- ever, SDS–PAGE analysis revealed that the peak B contained no collagenous protein. Together with these data, the present type V collagen preparation predominantly consisted of a molecule designated as [a1(V)]2a2(V), a most prevalent molecular form of type V collagen.
Compositional analyses, the type V collagen isolated from the porcine intestine by the
present procedure can be identified as the most prevalent [a1(V)]2 a2(V) form. Co-assembly of type XI collagen subunit was not detected in the present preparation.