FIGURE 5. Fractionation of P. aeruginosa OprF by gel filtration on Toyo Pearl HW-50F.

A, OprF preparation from P. aeruginosa PAO1 was fractionated on a high resolution column in the presence of 0.1% dodecyl maltoside and 0.4 m NaCl as described under “Experimental Procedures.” The leading edge of the OprF peak (seen from protein concentration in mg/ml (diamonds)) was seen to show a much higher specific poreforming activity (determined by the swelling rate of proteoliposomes upon dilution into iso-osmotic l-arabinose in the unit of ΔA₄₀₀/min/10 μg (triangles)) than the bulk of the peak. The total pore-forming activity per 10 μl of each fraction (in ΔA₄₀₀/min) is also shown (squares). The fact that the pore-forming activity of the leading edge is not due to other contaminating porin(s) is seen by the essentially undetectable pore-forming activity seen after the identical treatment of the fractions obtained from the outer membrane of an OprF-deleted strain, H636 (inverted triangles). B, portions (10 μl, after 5-fold dilution in sample buffer) of each fraction were separated by SDS-PAGE before or after heating at 100 °C in SDS-containing sample buffer of the samples, and the gel was stained with silver. Fraction 1 (the leading edge) is seen to contain significant portions of OprF behaving as oligomers (left panel, some of the bands are highlighted with arrowheads), which collapsed into a monomeric form after heat denaturation (right). The bands highlighted with arrowheads roughly correspond, in mobility, to 150, 70, and 55 kDa, respectively. Although several closely located bands are seen in most lanes, this is apparently due to the partial reduction of the two, closely spaced disulfide bonds in mercaptoethanol-containing running buffer as matrix-assisted laser desorption ionization time-of-flight analysis shows only OprF in each band.