We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

Eleven patients with chronic renal failure who were being treated with haemodialysis three times a week were monitored for a total of 34 haemodialysis sessions. Erythrocyte 2,3-biphosphoglycerate (2,3-BPG) concentration was analysed immediately before initiation of bicarbonate haemodialysis and 1 h afterwards. The 2,3-BPG concentration was expressed relative to the haemoglobin tetramer (Hb4) concentration as the 2,3-BPG/Hb4 ratio and compared with blood gas analyses and biochemical variables important for characterizing uraemia. During the first hour of haemodialysis the 2,3-BPG/Hb4 ratio decreased (p < 0.002), but the magnitude of the decrease did not significantly correlate with the 2,3-BPG/Hb4 ratio measured before haemodialysis (p=0.104). The decrease is most likely to be caused by the haemodialysis procedure itself. Mechanical stress on the erythrocytes is believed to cause the 2,3-BPG to escape; it is then removed by haemodialysis. Physiologically, an increase in 2,3-BPG would be expected to counteract the hypoxia which is frequently observed during haemodialysis. However, the present results show the opposite, a decrease in 2,3-BPG. No significant correlation was shown between the haemoglobin concentration and the 2,3-BPG/Hb4 ratio before dialysis (p=0.414). The pH showed a significant positive correlation with the 2,3-BPG/Hb4 ratio before dialysis, whereas the arterial pO2 and the 2,3-BPG/Hb4 ratio before dialysis were insignificantly negatively correlated. The concentrations of calcium, phosphate, creatinine, urea and albumin did not correlate significantly with the change in 2,3-BPG/Hb4-ratio after 1 h. The 2,3-BPG/Hb4 ratio (p=0.03) sampled just before dialysis correlated significantly and positively with the total weekly dosage of erythropoietin given to the patients.

Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations.  Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR.  These Safranines have two-electron midpoint potentials ranging from −272 to −315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties.  While this mixture may aid in Safranine O’s utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.

Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from −272 to −315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine O’s utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.

We are developing synthetic peptides that non-covalently attach to natural proteins, augmenting their properties and developing novel hybrid functional materials. Using a set of hydrophobic-residue containing collagen-mimetic peptides that self-assemble into nanodiscs, we aim to encapsulate membrane proteins, increase protein hydrogel hydrophobicity, and create novel materials using structural proteins. Preliminary results suggest nanodisc interactions with the reaction center-light harvesting complex I (RC-LH1) could elucidate additional RC-LH1 structural and functional information, useful for the development of next generation solar cells. Furthermore, electron micrographs of nanodiscs embedded in collagen type I (COL I) hydrogels provide evidence for the enhancement of hydrophobic properties of COL I, and the potential for sequestering hydrophobic molecules for drug delivery. Lastly, nanodisc induced assembly of the structural protein tropomyosin is explored with the intent of enhancing the structural properties of COL I hydrogels.