PubMed · 6434241
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1984. [Desk-top high frequency automatic centrifugal casting machine].. https://pubmed.ncbi.nlm.nih.gov/6434241/
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Promiscuous small molecules plague screening libraries and hit lists. Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes. It is not understood how aggregates inhibit their targets. To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregate-forming inhibitors with model proteins. By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact. This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein. beta-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction. Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates. This association could be reversed or prevented by the addition of Triton X-100. These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition. They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.
The effect of detergents and amphiphiles on protein solubility and their use in crystallization solutions was examined for an integral membrane protein, the light-harvesting I complex from Rhodospirillum centenum. Measurement by a centrifugation assay of the solubility of the protein in different detergents and amphiphiles showed high protein-solubility values when either octyl glucoside or lauryldimethylamine-N-oxide was present with heptanetriol or when deoxycholate was present with spermine. The detergent/amphiphile combinations that resulted in high protein solubility were shown to be successful for crystallization of the protein, suggesting that crystallization is favored for detergents and amphiphiles that optimize the solubility of integral membrane proteins. The amphiphiles effective for crystallization were found using laser mass spectrometry to displace the lauryldimethylamine-N-oxide bound to the protein. These results suggest that mass spectrometry can be used for screening of favorable crystallization conditions.
A dedicated CPC prototype permits direct flow pattern visualization in the partition cells of a CPC column. It was used to understand "flooding", a frequent phenomenon associated with large injections. A general strategy was developed to optimize the injection step in the framework of a particular preparative separation: the purification of 5-n-alkylresorcinols from a wheat bran lipid extract on a several hundred milligram scale. The construction of the "mobile phase/stationary phase/sample" pseudo ternary diagram characterizes the effect of the injected solution on mobile and stationary phases. The position of the binodal curve maximum indicates if the biphasic system is "robust" towards a large injection or not, and can be used for optimum mass load determination.