Direct, ultrasensitive, and selective optical detection of protein toxins using multivalent interactions.

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.

Effect of direct-fed fibrolytic enzymes on the lactational performance of dairy cows.

In trial 1, 30 midlactation (213 d in milk) Holstein cows were randomly assigned to a control or enzyme treatment in a two-period crossover design and were fed a total mixed ration based on alfalfa hay and silage. Cows on the enzyme treatment received an enzyme solution containing cellulases and xylanases, which was sprayed on the forage component of the ration at a rate of 1.65 ml/kg of forage dry matter (DM) between 8 and 24 h prior to feeding. Cows consuming the forage treated with enzyme produced more milk (27.2 vs. 25.9 kg/d) and digested more DM per day than did cows fed the control forage. In trial 2, 40 early lactation Holstein cows were assigned to one of four treatments for 16 wk. Following a 2-wk covariate period, cows were assigned to 1) no enzyme treatment, 2) a low (1.25 ml/kg of forage DM) enzyme treatment, 3) a medium (2.5 ml/kg of forage DM) enzyme treatment, or 4) a high (5.0 ml/kg of forage DM) enzyme treatment. Enzymes were a 2:1 combination of cellulase and xylanase diluted in water and sprayed on a combination of alfalfa hay and silage and whole cottonseed immediately before mixing with a concentrate based on barley. Dry matter intakes were similar for cows on treatments 2, 3, and 4 and were greater than those for cows on treatment 1. Production of milk, 3.5% fat-corrected milk, and energy-corrected milk was greater for cows on treatment 3 than for cows on treatment 1. Fibrolytic enzymes applied to the forage portion of the rations prior to feeding improved lactational performance of early and midlactation cows.

Integrated optical biosensor for detection of multivalent proteins.

We have developed a simple, highly sensitive and specific optical waveguide sensor for the detection of multivalent proteins. The optical biosensor is based on optically tagged glycolipid receptors embedded within a fluid phospholipid bilayer membrane formed upon the surface of a planar optical waveguide. Binding of multivalent cholera toxin triggers a fluorescence resonance energy transfer that results in a two-color optical change that is monitored by measurement of emitted luminescence above the waveguide surface. The sensor approach is highly sensitive and specific and requires no additional reagents and washing steps. Demonstration of protein-receptor recognition by use of planar optical waveguides provides a path forward for the development of fieldable miniaturized biosensor arrays.

The effects of high pressure upon ligated and deoxyhemoglobins and myoglobin. An optical spectroscopic study.

The effects of high pressure (0.1-3.4 gigapascal (GPa)) on the ferrous heme active sites of human adult hemoglobin, sperm whale myoglobin, and Glycera dibranchiata hemoglobin (Fraction II) were probed using resonance Raman and absorption spectroscopies. High-to-low spin transitions of the heme iron occur for hemoglobin, myoglobin, and Glycera hemoglobin at 0.35, 0.75, and 0.50 GPa, respectively, for the deoxy species. These interspecies differences result from variations in the composition of the hemepockets and/or their rigidity to pressure-induced volume changes. Heme active sites initially bound to CO or O2 exhibit distinctive behavior at high pressures. For all proteins studied, O2 apparently dissociates from the heme at only moderately high pressure, while CO remains bound to the heme moiety even at extreme pressures. The Raman spectra demonstrate the differences in the ligated and deoxy species at 3.4 GPa in the high frequency region. Discrete changes (i.e. iron spin-state transitions and dissociation of O2) occur that are commensurate with the collapse of the distal pocket, while continuous shifts in the absorption and Raman spectra are observed at pressures above those required for pocket collapse.

Temperature dependence of the resonance Raman spectra of plastocyanin and azurin between cryogenic and ambient conditions.

Resonance Raman spectra of spinach plastocyanin and Pseudomonas aeruginosa azurin were studied as a function of temperature between 10 K and 300 K. The spectra are markedly improved both in signal/noise ratio and in resolution at low temperatures. The assignments of the resonance Raman-active vibrations are reinterpreted in view of the number and intensities of peaks observed in the low-temperature spectra. Features appear in the low-temperature spectra of azurin that may be due to copper-bound methionine. The plastocyanin spectra undergo a transition between 220 K and the melting point of water that results in dramatically narrowed peaks at lower temperature and a shift in the carbon-sulfur stretching frequency of the copper-bound cysteine, suggesting a structural change in the active site and an accompanying effect on vibrational dephasing. Considering that the structures and nonvibrational spectroscopies of these two proteins are similar, the substantial differences in the resonance Raman spectra are striking and significant.