Thermal (in)stability of type I collagen fibrils.

We measured the Young's modulus at temperatures ranging from 20 to 100 degrees C for a collagen fibril that is taken from a rat's tendon. The hydration change under heating and the damping decrement were measured as well. At physiological temperatures 25 to 45 degrees C, the Young's modulus decreases, which can be interpreted as an instability of the collagen. For temperatures between 45 and 80 degrees C, the Young's modulus first stabilizes and then increases when the temperature is increased. The hydrated water content and the damping decrement have strong maximums in the interval 70 to 80 degrees C indicating complex intermolecular structural changes in the fibril. All these effects disappear after heat-denaturation of the sample at 120 degrees C. Our main achievement is a five-stage mechanism by which the instability of a single collagen at physiological temperatures is compensated by the interaction between collagen molecules.

Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection.

The aim of the present work is to develop an evanescence wave array biosensor exploiting the "kinetic" approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO(2) layer to increase the surface area available for immobilization. The biosensor enabled the detection of 2.5 microM paraoxon, and 10 microM DFP and parathion, respectively. Very short response time of 30s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 degrees C, the waveguide retained reasonable activity for greater than 45 days.

The influence of external environment fluctuations on the signal formation of microbiosensors.

This part of theoretical analysis describes the fluctuations of output signal of microbiosensors when the number of accessible molecular recognition elements (enzymes, receptors, antibodies, etc.) fluctuated under external environmental influences. The mean electric current, dispersion correlating function, as well as spectral density of output current fluctuation are analyzed, and it is shown that a comparison of theoretically calculated mean current and correlation function with experimental data allow a determination of the kinetic parameters of substrate binding reaction with the molecular recognition element of biosensor.

Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins.

A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.

Enhancement of the specificity of an enzyme-based biosensor for L-tryptophan.

A new selective amperometric biosensor for reagentless L-tryptophan determination has been developed using immobilized tryptophan-2-monooxygenase (TMO, EC 1.13.12.3). This enzyme-based biosensor provides a rapid-response detection system for concentrations of L-tryptophan between 25 and 1,000 microM in a batch mode system and between 100 and 50,000 microM in a flow-injection mode. The response time was 30 seconds, and the total analysis time was less than 3 minutes. The biosensor retained catalytic activity and fidelity of phenylalanine and tryptophan response for greater than 4 months with repeated usage. The biosensor selectivity to L-tryptophan was dramatically increased relative to phenylalanine when a competitive inhibitor of TMO, indole acetamide (IA), was included. The biosensor was successfully used for L-tryptophan determination in nutrition broth, giving values identical to those determined by HPLC analysis.

A tryptophan-2-monooxygenase based amperometric biosensor for L-tryptophan determination: use of a competitive inhibitor as a tool for selectivity increase.

A new flow-injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L-tryptophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve between 0.1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25 degrees C and stored at 8 degrees C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 microM IA, the biosensor response to L-phenylalanine decreased to 7-4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritional broth.

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins.

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

L-Lysine-2-monooxygenase production by strains of Pseudomonas fluorescens and P. putida.

Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained L-lysine as the only source of carbon and nitrogen, and screened for L-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.

A biosensor for L-proline determination by use of immobilized microbial cells.

A biosensor to quantify L-proline within 10(-5)-10(-3) mole/L concentration is described. Immobilized Pseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.

[Isolation, purification and various properties of L-lysine-2-monooxygenase from Pseudomonas putida]. / Vydelenie, ochistka i nekotorye kharakteristiki L-lizin-2-monooksigenazy iz Pseudomonas putida.

Isolation and purification of L-lysine-2- monooxygenase from the bacterium Pseudomonas species was carried out. The purification procedure included ammonium sulfate fractionation, acid treatment, gel filtration through Sephadex G-200 and ion-exchange chromatography on DEAE-Sephadex A-50. Such treatment resulted in more than 220-fold purification and 22% yield; the specific activity of the enzyme is 14.6 U/mg. The enzyme spectrum is typical for flavoproteins, with peaks at 275, 386 and 462 nm. At 460 nm excitation, the enzyme fluorescence has an emission maximum at 530 nm, whereas at 360 nm extication--at about 520 nm. The molecular mass of L-lysine-2-monooxygenase as determined by SDS/PAAG electrophoresis is about 268 kD. The KM values for oxygen and lysine are equal to 6.5.10(-4) M and 2.3.10(-4) M, respectively. The curve for the dependence of the reaction rate on lysine concentration is sigmoidal. It was assumed that the electrophoretic behaviour of the enzyme confirms the hypothesis on the nature of allosteric regulation of the enzyme activity by alterations in the regulatory site charge.

[Sodium currents in muscles incubated in potassium-free Ringer's solution. Effect of ouabain on unidirectional sodium currents]. / Potoki natriia v myshtsakh, inkubirovannykh v beskalievom rastvore Ringera. Vliianie uabaina na odnonapravlennye potoki natriia.

Frog sartorius muscles subjected to loading with Na in K-free Ringer solution in the cold were subsequently labelled with 22Na. The uptake of 22Na is not sensitive to ouabain (10(-4) M) while sodium efflux is decreased by oubain. It is concluded that ouabain-sensitive Na-for Na interchange is not present in this condition. Possibly ouabain-sensitive sodium efflux is partly or completely potassium-requiring fraction since some K (approximately 10 microM) is inevitably present in K-free solution. The increase in the rate constant for potassium loss in the presence of ouabain favours this supposition.

[Relationship between unidirectional sodium ion currents and their concentration in muscle and medium]. / Zavisimost' odnonapravlennykh potokov ionov natriia ot ikh soderzhaniia v myshtse i srede.

Sodium fluxes were measured as concentrations of external sodium and intracellular sodium were changed under K-free condition. Paired sartorious muscles with high intracellular sodium concentration from frog (Rana ridibunda) were used throughout this investigation. The measured uptake of 22Na (mM/kg . h) is directly proportional to intracellular sodium but when influx of 22Na is calculated per unit surface (pmole/cm2s) taking account of "effect of relation volume/surface" the influx is to be independent of Nain. Sodium influx varied linearly with changing external Na. Sodium efflux is not dependent on external sodium concentration. The results are in favour of the conclusion that the sodium fluxes across the skeletal muscle membrane do not exhibit the mechanism of sodium--sodium exchange.