The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate~s! and trapping the folding intermediate~s! into enzymatically inactive, "aggregation-insensitive" state~s!. However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations~Ͼ1.5 M!, proline is shown to form loose, higher-order molecular aggregate~s!. The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.
An attempt to tune the electronic properties of pyrene (Py) by coupling it with a strong electron donor (-PhNMe2, DMA)/acceptor (anthronitrile, AN) through an ethynyl bridge has been undertaken. A moderate electron donor (iPrOPh-, IPP)/acceptor (2-quinolinyl, 2Q) has also been incorporated, and all four molecules were studied with reference to a neutral molecule, namely, 1-phenylethynylpyrene (PhEPy). All the arylethynylpyrenes (ArEPy's) have been thoroughly characterized, and their electronic properties were studied by absorption and emission spectral properties of these ArEPy's. The electrochemical characteristics were also studied for arriving at the electrochemical band gap which has been compared with the HOMO-LUMO energy gap derived from the photophysical measurements and theoretical calculations performed by density functional theory (DFT) using B3LYP/6-31G basis sets. The results obtained from experimental and theoretical studies are critically discussed.
A stable, covalent, and highly active protein microarray was created through the formation of cyclic esters between surface boronic acids and the carbohydrate moiety of a fusion protein, Fc‐dectin‐1 (see picture). A biotin‐labeled polysaccharide was used as a probe to investigate the binding activity of the protein. Staining of the product array with streptavidin‐Cy3 revealed the effectiveness of this immobilization strategy.
We have developed a sensitive electrochemical immunoassay system for the detection of a protein tumor marker, carcinoembryonic antigen (CEA), that is based on a carbon nanoparticle (CNP)/poly(ethylene imine) (PEI)-modified screen-printed graphite electrode (CNP-PEI/SPGE) covered with anti-CEA antibodies. The signal amplification strategy--using CdS nanocrystals as biotracers and CNPs to enhance electron transfer--improves the sensitivity and detection limit for CEA, suggesting that this system holds promise for development into a point-of-care or disposable home-care self-diagnostic tool. This biosensor is based on a sandwich complex immunoassay, which we assembled from sequential layers of the anti-CEA antibody (alphaCEA) on CNP-PEI/SPGE, the CEA sample, and the CdS nanocrystal quantum dots (QDs) sensitized with alphaCEA (alphaCEA-CdS QD). We used square wave anodic stripping voltammetry (SWASV) to amplify the signal current response obtained from the dissolved alphaCEA-CdS QDs. The calibration curve for CEA concentration was linear in the range of 0.032-10 ng/mL; the detection limit (estimated as the mean of the blank sample plus three times the standard deviation obtained on the blank sample) was 32 pg/mL (equivalent to 160 fg in a 5 microL sample). This method is suitably precise and sensitive to function as a means of determining urinary CEA, which is a better marker than serum CEA for the early detection of urothelial carcinoma.
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