X-ray fluorescence used to characterize the salt content of proteins

The quantitative measurement of the salt content in solid protein samples was performed using X-ray fluorescence. Linear calibration curves were obtained for chloride, calcium and sulfur using sulfur and chloride as internal standards in the range 1-10 protein molar equivalents. The detection limit was approximately 0.02 molar equivalents for chloride and less than 0.01 molar equivalents for calcium. X-ray fluorescence thus provides a non-destructive sensitive method of testing the efficiency of different purification methods. Commercial hen egg white lysozyme samples contain from 15 to 46 molar equivalents of chloride, whereas the calcium content remains less than 0.2 equivalents. Deionization on ion-exchange resins is a very efficient tool for removing ionic species since deionized lysozyme samples contain less than 0.34 molar equivalents of chloride. Extensive dialysis against water only partially removes chloride ions, the residual chloride content corresponding to the number of counter-ions necessary to ensure the electroneutrality of lysozyme when dissolved in water.