Effect of the mitochondrial membrane potential on the absorbance of the reduced form of cytochrome c oxidase.

The effect of mitochondrial membrane potential (ΔΨ) on the absorbance of the reduced cytochrome c oxidase (COX) was evaluated in isolated rabbit heart mitochondria using integrating sphere optical spectroscopy. Maximal reduction of the mitochondrial cytochromes was achieved by either blowing nitrogen to remove oxygen, or by adding cyanide. Gradual depolarization of ΔΨ by adding increasing concentrations of uncoupler resulted in an increase of up to 50 % in the absorbance of cytochrome aa under nitrogen saturation, and of 25 % with cyanide.

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria.

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a-Cu binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420-450 nm), alpha (560-630 nm) and red (630-700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme.

Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs.

Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq.