Skip to main content
. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Free Radic Biol Med. 2015 Jul 6;89:83–90. doi: 10.1016/j.freeradbiomed.2015.07.001

Figure 5.

Figure 5

Effect of HOCl treatment on folding and structure of NC1 hexamer of collagen IV isolated from rat kidneys. (Upper panel) Purified NC1 hexamers were incubated in 50 mM TBS, pH 7.4 (solid line) or in the same buffer supplemented either with 50 μM HOCl (dotted line) or with 50 μM HOCl and 100 μM PM (dashed line) for 2 h at 37°C. Final protein concentration was 0.2 mg/mL. Samples were then supplemented with 6 M GdnCl and denatured at 80°C for 30 min. Samples were injected onto gel-filtration FPLC column equilibrated with 50 mM TBS to allow for NC1 domain refolding and hexamer reassembly and analyzed as described under Experimental procedures. (Lower panel) Purified NC1 hexamers (1 mg/mL) were incubated in 50 mM TBS, pH 7.5 alone, with 1 mM HOCl or with 1 mM HOCl and 1 mM PM for 2 h at 37°C. Samples were washed and concentrated to 0.8 mg/mL NC1 hexamer. Samples were then subjected to a limited proteolysis with proteinase K (PrK) as described under Experimental procedures. Samples (8 μg of NC1) were fractionated on 12% SDS-PAGE followed by Coomassie Brilliant Blue staining.