Anomalous behavior of bovine αs1- and β-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Abstract

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of M r and the mobility of the protein through the gel matrix. However, bovine caseins (especially α s1-casein) have lower mobilities than expected on the basis of their known M r. The binding of SDS to both α s1-casein ( M r 23,600) and β-casein ( M r 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the α s1-casein:SDS complex was larger than the β-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both α s1- and β-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to α s1- and β-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of α s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected β-casein mobility. These and other results indicate that the low electrophoretic velocity of α s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of α s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the α s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the M r of the protein.