I n the quest for novel pharmaceuticals to treat important diseases, biologists often use in vitro assays to assess apparent activity. Thus, when testing novel synthetic compounds in search of a new class of drugs, it is important to know the solubility of the compound under the assay conditions used so that the activity data may be interpreted correctly. As the screening throughput increases, so do the demands on solubility determinations and the reliance on this data to aid in the selection of the more promising compounds for further studies. Since many compounds synthesized in drug discovery compounds contain UV chromophores, solubility determinations using UV plate readers are widely applicable to common druglike compounds. A more analytically rigorous approach calculates the solubility using a calibration curve rather than a ratio to a reference concentration. Rather than use the l max of the analyte for quantification, an automated method is used for intelligent selection of the wavelength most appropriate for quantification. Using a BioMek FX and Peak Seeker, our visual basic application written in-house, 128 compounds may be assayed at a single pH within 5 h using MultiScreen Solubility plates.
The static infrared absorption (IR) and circular dichroism (CD) spectra of the temperature-dependent conformational states of apomyoglobin in potassium phosphate and sodium acetate buffer solution at pH 5.3 in D2O are reported. The circular dichroism ellipticity at 222 nm reveals a loss of helical structure for both heat- and cold-denatured states. Cold denaturation leads to a molten globule state with an associated partial loss of helical structure identified at 1646 cm−1. Heat denaturation also exhibits an absorption loss at 1646 cm−1 but leads to the appearance of aggregation at 1680 and 1619 cm−1. There is semi-quantitative agreement of the ultraviolet circular dichroism and infrared estimates of protein helicity. The denaturation spectra give evidence that there are specific associations between acetate ions and the protein, but not between phosphate ions and the protein.
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