Characterizing osmolyte chemical class hierarchies and functional group requirements for thermal stabilization of proteins.

Osmolytes are naturally occurring organic compounds that protect cellular proteins and other macromolecules against various forms of stress including temperature extremes. While biological studies have correlated the accumulation of certain classes of osmolytes with specific forms of stress, including thermal stress, it remains unclear whether or not these observations reflect an intrinsic chemical class hierarchy amongst the osmolytes with respect to effects on protein stability. In addition, very little is known in regards to the molecular elements of the osmolytes themselves that are essential for their functions. In this study, we use differential scanning fluorimetry to quantify the thermal stabilizing effects of members from each of the three main classes of protecting osmolytes on two model protein systems, C-reactive protein and tumor necrosis factor alpha. Our data reveals the absence of a strict chemical class hierarchy amongst the osmolytes with respect to protein thermal stabilization, and indicates differential responses of these proteins to certain osmolytes. In the second part of this investigation we dissected the molecular elements of amino acid osmolytes required for thermal stabilization of myoglobin and C-reactive protein. We show that the complete amino acid zwitterion is required for thermal stabilization of myoglobin, whereas removal of the osmolyte amino group does not diminish stabilizing effects on C-reactive protein. These disparate responses of proteins to osmolytes and other small molecules are consistent with previous observations that osmolyte effects on protein stability are protein-specific. Moreover, the data reported in this study support the view that osmolyte effects cannot be fully explained by considering only the solvent accessibility of the polypeptide backbone in the native and denatured states, and corroborate the need for more complex models that take into account the entire protein fabric.

Determination of B, Si, Cr, Mo, Th and Hf in UF(6) by end-on viewed ICP-AES.

A spectrographic analysis method is described for the determination of B, Si, Cr, Mo, Th and Hf in UF(6) Uranium is separated from UO(2)F(2) using a TBP [tributyl phosphate] extraction-elution resin column technique. The six impurity elements, including B and Si, in the fluorine-containing extraction-elution solution are determined by end-on viewed ICP-AES. The method is simple, rapid and suitable for routine analysis.