Calculation of protein extinction coefficients from amino acid sequence data.
ABSTRACT Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.
- SourceAvailable from: Marcela Cristina De Moraes[Show abstract] [Hide abstract]
ABSTRACT: Prion diseases are characterized by protein aggregation and neurodegeneration. Conversion of the native prion protein (PrPC) into the abnormal scrapie PrP isoform (PrPSc), which undergoes aggregation and can eventually form amyloid fibrils, is a critical step leading to the characteristic pathomorphologial hallmark of these diseases. However, the mechanism of conversion remains unclear. It is known that ligands can act as cofactors or inhibitors in the conversion mechanism of PrPC into PrPSc. Within this context, herein, we describe the immobilization of PrPC onto the surface of magnetic beads and the morphological characterization of PrPC-coated beads by fluorescence confocal microscopy. PrPC-coated magnetic beads were used to identify ligands from a mixture of compounds, which were monitored by UHPLC-ESI-MS/MS. This affinity-based method allowed the isolation of the anti-prion compound quinacrine, an inhibitor of PrP aggregation. The results indicate that this approach can be applied to not only “fish” for anti-prion compounds from complex matrixes, but also to screening for and identify possible cellular cofactors involved in the deflagration of prion diseases.Journal of Chromatography A 12/2014; http://dx.doi.org/10.1016/j.chroma.2014.12.014. · 4.26 Impact Factor
- Frontiers in Physiology 12/2014; 5(465):1-11.
- [Show abstract] [Hide abstract]
ABSTRACT: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with KD values in a μM range. This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.Frontiers in physiology. 01/2014; 5:465.