(A) Sedimentation
equilibrium experiments with SwHaCE in solution.
Using rotor speeds of 9000, 14000, and 18000 rpm
(which are equal to 6500g, 15800g, and 26100g, respectively), and following protein
at 280 nm, the calculated molecular mass of SwHaCE
is 144.3 kDa, indicating a dimer in solution at equilibrium. (B) Sedimentation
equilibrium experiment for SwHaCE in solution with
the SwH-NOX Fe(II)–NO complex. Using rotor
speeds of 9000, 14000, and 18000 rpm (which are equal to 6500g, 15800g, and 26100g,
respectively), and following protein complexes at 400 nm, the calculated
molecular mass for the complex is 197.2 kDa at equilibrium. This matches
the molecular mass expected for a heterotetramer of two SwH-NOX proteins and two SwHaCE proteins. Each experiment
was conducted in triplicate.