FIGURE 5.

Molar protein mass of Tween 20-solubilized H⁺,K⁺-ATPase after size exclusion chromatography with registration of light absorbance at 280 nm, refractive index, and light scattering. Shown is a superposition of A₂₈₀ (green, thin), differential refractive index (blue, thin), and light scattering (red) signals, in arbitrary units (left axis), and molar masses for the glycosylated protein (blue, thick), detergent + lipid (green, thick), and complex (i.e. their sum (purple) (right axis)). The calculation considers (∂n/∂c) for the glycosylated protein to be 0.185 ml/g (with 9 kDa of carbohydrate for a monomeric α,β protein) and (∂n/∂c) for detergent + lipid to be 0.082 ml/g, with extinction coefficients of 0.975 and 0.0115 ml/(mg cm), respectively. H⁺,K⁺-ATPase after solubilization was subjected to size exclusion chromatography on a Superose 6 column; fractions corresponding to the central part of the main protein peak were pooled, and 30 μl were injected onto a KW 804 column equilibrated at 4 °C, with a flow rate of 0.5 ml/min, with an eluent filtered at 0.1 μm and containing 40 mm KCl, 40 mm MES (pH 6), 0.25 m sucrose, 3 mm MgCl₂, 1 mm EDTA, 2 mg/ml Tween 20.