Polymers as surface-based tethers with photolytic triggers enabling laser-induced release/desorption of covalently bound molecules.

A novel design for surface-based macromolecular docking and release is presented together with a strategy to improve and extend biopolymer structure determination capabilities. Polymeric surfaces with arrays of tethers for covalent molecular attachment were designed with photolytic triggers to enable spatially defined, laser-induced uncoupling/desorption of the tethered molecules. Upon photolytic cleavage, a defined portion of the tether ("tail") remains attached to the biomolecule as a probe. Chemically defined memory, determined by the number of reporter tails, reflects the biomolecule interaction with tether-probe devices encountered (i.e., footprint) on the probe surface. To demonstrate function, a surface of poly(4-vinylpyridine) was extended through the pyridinium nitrogens with spacer arms (-N-ethylsuccinamyl-) producing photolytic pyridinium nitrogen bonds. The photolabile tether was terminated with leaving groups (N-hydroxysulfosuccinimide) for covalent attachment of biopolymers. An 18-residue peptide (N terminus of human beta-casein) was covalently docked to these tether-probes, irradiated with coherent UV light, and released with two reporter tails of a mass predicted by tether formation at the two primary amine groups and subsequent photolytic cleavage at the intended site. This is the first demonstration of polymeric surface structure enabling the covalent docking and laser-induced uncoupling/desorption of intact macromolecules through the use of photolytic tethers. Surface-based tether-probe devices, operated by coherent light, should advance our ability to explore covalent modifications in biopolymer structure and alterations in conformation, generated either in advance of tethering or through chemical/enzymatic manipulations performed directly in situ.

[Kinetic study of 3,4-dihydroxyphenyl-L-alanine synthesis in Citrobacter freundii cells immobilized in carrageenan]. / Kineticheskoe izuchenie sinteza 3,4-dioksifenil-L-alanina kletkami Citrobacter freundii, immobilizovannymi v karraginan.

A kinetic study was carried out of the enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) by the Citrobacter freundii 62 cells, possessing tyrosine-phenol-lyase (TPL) activity, immobilized in carrageenan, and optimum conditions of the reaction were found. The dependence of the TPL activity and its stability on the conditions of the DOPA synthesis was investigated. The TPL activity was higher and more stable in the immobilized cells as compared to free ones.

[Enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine by free and immobilized Citrobacter freundii cells]. / Fermentativnyi sintez 3,4-dioksifenil-L-alanina svobodnymi i immobilizovannymi kletkami Citrobacter freundii.

The Citrobacter freundii 62 cells immobilized in PAAG and possessing the tyrosine-phenol-lyase (TPL) activity catalyse the synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) from pyrocatechol and ammonium pyruvate. The synthesis of DOPA was studied using both free and immobilized bacterial cells. When the concentration of pyrocatechol is over 0.1 M the TPL activity of the cells is inhibited. The concentration of pyrocatechol can be increased up to 0.3 M by using an equimolar mixture of pyrocatechol and boric acid. The addition of ascorbic acid as an antioxidant results in a lower TPL activity of both free and immobilized bacterial cells.