alpha-chymotrypsin and methyl-iodide

The interface between nematic liquid crystal, 4-cyano-4'-pentylbiphenyl (5CB), and water in a transmission electron microscopy (TEM) grid cell coated with QP4VP-b-LCP (quaternized poly(4-vinylpyridine) (QP4VP) and poly(4-cyanobiphenyl-4'-oxyundecylacrylate) (LCP)) was examined for protein and DNA detection. QP4VP-b-LCP was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Quaternization of P4VP with iodomethane (CH3I) made it a strong cationic polyelectrolyte and allowed QP4VP-b-LCP to form complexes with oppositely charged biological species. Several proteins, such as bovine serum albumin (BSA), hemoglobin (Hb), α chymotrypsinogen-A (ChTg), and lysozyme (LYZ), were tested for nonspecific protein detection. By injecting the protein solutions into the TEM grid cell, the initial homeotropic orientation of the TEM grid cell changed to a planar orientation above their isoelectric points (PIs) due to electrostatic interactions between QP4VP (+charge) and proteins (-charge), which did not occur below the PIs of the tested proteins. Their minimum concentrations at which the homeotropic to planar configurational change (H-P change) occurred were 0.01, 0.02, 0.03, and 0.04 wt.% for BSA, ChTg, Hb, and LYZ, respectively. One of the strong anionic polyelectrolytes, deoxyribonucleic acid (DNA) (due to the phosphate deoxyribose backbone) was also tested. A H-P change was observed with as little as 0.0013 wt.% salmon sperm DNA regardless of the pH of the cell. A H-P change occurred in 5CB and was observed by polarized optical microscopy. This simple and inexpensive setup for nonspecific biomaterial detection provides the basic idea for developing effective selective biosensors by introducing specific binding groups, such as the aptamer and antibody.

Topics: Animals; Biosensing Techniques; Biphenyl Compounds; Chymotrypsin; DNA; Electrolytes; Hemoglobins; Hydrocarbons, Iodinated; Isoelectric Point; Liquid Crystals; Magnetic Resonance Spectroscopy; Microscopy, Electron, Transmission; Muramidase; Nitriles; Optics and Photonics; Polymers; Polyvinyls; Salmon; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Static Electricity

Lyophilized proteins were reacted in vacuo with a volatile reagent or dispersed in octane and reacted with dissolved reagent. Three novel derivatives were formed with iodomethane: (a) quaternized trimethyl amino groups, (b) N1,N3-dimethylimidazolium cation, and (c) phenolic O-methyl ether. Acid anhydrides acylated amino groups and formed mixed anhydrides with side-chain carboxyl groups. Under nonaqueous conditions it was observed that: (i) The same derivatives are formed as under aqueous conditions. (ii) Hydrolytic breakdown of protein is prevented. (iii) Less reagent is required. (iv) Unreacted reagent can be recovered. (v) Water-labile derivatives can be isolated as stable intermediates. (vi) The yield of a derivatized functional group was directly related to its pK(a), its surface exposure, and the pH of the solution from which the protein was lyophilized. (vii) The physicochemical factors governing the reactivity of protein functional groups in nonaqueous environments appear to reflect the protein solution structure prior to lyophilization.

Topics: Acetic Anhydrides; Acylation; Animals; Cattle; Chymotrypsin; Freeze Drying; Hydrocarbons, Iodinated; Hydrolysis; Insulin; Magnetic Resonance Spectroscopy; Methylation; Protein Conformation; Proteins; Ribonucleases; Serum Albumin, Bovine