Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin.

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.