Capillary electrophoresis of S. nuclease mutants.
The electrophoretic migration behavior of 12 S. nuclease variants from Staphylococcus aureus with small but well defined structural differences from site directed mutation was investigated in free solution capillary electrophoresis at pH 2.8 to 9.5. The nucleases are basic proteins; the pI and the M(r) of the wild type are 10.3 and 16.811 kd, respectively. With specially selected oligoamino buffers and with an inert, hydrophilic wall coating in 75 microns I.D. quartz capillary tubes, most of the proteins could be separated by CZE without interference by wall adsorption even at pH 9.5 where the selectivity was the highest. At pH 2.8, 4.1 and 7.0, S. nucleases are known to be in the random coil, "swollen" and the tight native state. Assuming that in a given state, i.e., at a certain pH, the molecular radii of the nucleases are the same, their hydrodynamic radii were calculated from their pertinent electrophoretic mobilities. The respective radii of 50.1, 26.8, and 25.0 Angstrum thus obtained agreed very well with the corresponding radii of gyration obtained from X-ray scattering. In fact, from the electrophoretic mobilities at pH 9.5, the existence of a hitherto unknown swollen basic state of the nuclease having a hydrodynamic radius of 30.5 Angstrum was postulated. In addition, a method was described to evaluate the valence of the protein at different pH from their pertinent electrophoretic mobilities. A general advantage of this method is that only the differences between the valences of the mutants and the wild type are needed; and for none of the proteins is required the knowledge of the actual valence. The results of the methods allowed the construction of a pH profile of the protein's valence. For the wild type, this profile was compared to the H+ titration curve and the agreement was excellent. Both methods employed some novel structure-electrophoretic mobility relationships and the predicted protein properties compared remarkably well to the values obtained by exoelectrophoretic methods such as pH titration and X-ray scattering. Surprisingly, certain S. nucleases having the same valence could also be readily separated by CZE in some cases under the same conditions used for the others. Close examination of appropriate X-ray crystallography and/or NMR data indicated subtle differences in the molecular structure of these proteins that could be responsible for slight alteration in their hydrodynamic radii.(ABSTRACT TRUNCATED AT 400 WORDS)
Kálmán
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Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA.
Ma
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Fox
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Horváth
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D000595:Amino Acid Sequence; D002021:Buffers; D004586:Electrophoresis / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D013211:Staphylococcus aureus / Q000201:enzymology*
D002021:Buffers; D008836:Micrococcal Nuclease