Unexpected mobility of human lambda chains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Abstract

Molecular weight determination of human immunoglobulin heavy chains through sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been successfully accomplished and reported (Virella and Parkhouse, 1972, 1973a). When the same electrophoretic system was applied to light chain mol. wt estimation, unexpected differences in the mobilities of kappa and lambda chains were found. Normal human IgG (Cohn's Fr. II), kindly provided by Soc. Com. Multiradix, Lisbon, and 24 isolated monoclonal proteins (8 IgGK, 8 IgGL, 2 IgAK, 2 IgMK and 2 IgML) were used in this study as source of light chains. Proteins were reduced in the following conditions: (a) Room temperature reduction of proteins diluted in phosphate buffered saline containing 100 mM dithioerythritol, followed, after 60 min, by alkylation at 0°C with iodoacetamide (50~o molar excess over sulphydryl). (b) Reduction under alternating periods of heating to 100°C and room temperature incubation of proteins diluted in phosphate buffer pH 7.2, 0.01 M, containing 2~ (w/v) sodium dodecyl sulphate and 50 mM dithioerythritol. Under these circumstances all disulphide and non-covalent bonds appear to be eliminated, and mol. wt determinations become quite accurate (Virella and Parkhouse, 1972, 1973a). The component chains of the reduction mixture were separated electrophoretically in a sodium dodecyl sulphate polyacrylamidc gel system (Summers et al., 1965). Gel cylinders measured 65 × 6mm, and acrylamide concentrations were of 5 and 75~o (w/v). All samples were heated to 100°C immediately before being submitted to electrophoresis, in order to ensure absence of aggregation (Virella and Parkhouse, 1973a, b). Molecular weights were calculated according to Weber and Osborn (1969). Different control proteins were employed for each reduction method (Table 1) being subjected to the same reducing conditions as immunoglobulins. For calibration purposes the mobility of light chains was assumed to be that of the more intensely stained and fastermigrating protein fraction when two were separated on the light chain region. As shown by Fig. 1, kappa and lambda chains separated from four 100°C-reduced monoclonal proteins typed as IgG had different mobilities in 5~ (w/v) polyacrylamide gel. The same differences can be observed in Fig. 2, where the separation of four room temperature-reduced monoclonal proteins, typed as IgA and IgM, is illustrated. This last figure also shows that the light chain region from normal IgG reduced under the same conditions is constituted by two protein bands of unequal staining intensity. The nature of those two light chain fractions in normal IgG was established immunochemically by the method described by Virella and Parkhouse (1971), as shown in Fig. 3. The upper and less intensely stained band was constituted