CHARGE AND SIZE EFFECTS IN THE CAPILLARY ZONE ELECTROPHORESIS OF NUCLEASE-A AND ITS VARIANTS

The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investiga ted in the light of their three-dimensional structure known from X-ray crystallography and nuclear magnetic resonance (NMR) measurements. Nu clease A (molecular mass 16.8 kDa, pK(a) 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal s tructure. With the use of coated quartz capillaries and suitable buffe rs, the protein migration was investigated at pH from 2.8 to 9.5 witho ut interference by wall adsorption. Although the selectivity of the el ectrophoretic system for the proteins was mainly determined by their c harge differences, certain variants having the same net charge could a lso be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K1 16G so that they could be separated by CZE. The structures of both var iants are the same except for the solvent-exposed loop containing resi due 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 t o 117 amino acids protrudes slightly from the protein, with a concomit ant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its la rger hydrodynamic radius despite its smaller molecular mass. The inter pretation of the experimentally measured mobilities of such closely re lated proteins therefore requires not only consideration of their elec trostatic charge but also the fine details of their molecular structur es.