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. This concern seems to be substantiated by in vitro studies (34) showing the proinflammatory effect of lysed neutrophils in an elastase-dependent manner
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. Such a link between the instability to freeze-thawing and propensity to bind ANS in ice supports the notion that irreversible damage to freeze labile proteins, often resulting in enzyme inactivation and or protein aggregation, is likely to have originated from partial unfolding of the globular structure in ice
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. Because the spectrum reflects the hydrophobicity of the binding site, shifting to the blue in less polar environments (30,31), the above correlation suggests that extensive binding of ANS is associated with the exposure of hydrophobic regions of the macromolecule that are generally buried and inaccessible to the probe
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. In our previous titration experiment on the CaM:SEF2-1mp complex (11) we always observed a single set of CaM resonances that shifts position upon different CaM:SEF2-1mp ratios, i
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. For a molecule existing in two different states but with similar atom-positional fluctuations, similar configurational entropies are expected for the two states
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. The sensitivity of the ANS binding method to the tertiary structure of proteins in ice should find practical utility in devising safer freeze-drying protocols of pharmaceutical proteins and testing the effectiveness of various stabilizing/ destabilizing agents employed in protein formulations
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