Glossoscolex paulistus hemoglobin structure is composed of 144 globin chains and 36 polypeptide chains lacking the heme group, with a total molecular mass of 3600 kDa. The current study focuses on ...the oxy-HbGp oligomeric stability, as a function of the storage time, at pH 7.0, using dynamic light scattering, analytical ultracentrifugation (AUC), optical absorption and size exclusion chromatography (SEC). HbGp stored in Tris-HCl buffer, pH 7.0, at 4 °C, for two years remains in the native form, while 4–6 years HbGp stocks present typical hemichrome species absorption spectra. AUC and SEC analyses show that the contribution of HbGp-subunits, such as, dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d, increases with the protein aging due to the lower stability of the HbGp with the time. The dissociation and the oxidation of the iron noted for the older protein solutions indicate that HbGp storage for periods of time longer than two years changes its ability to carry oxygen. Despite the reduction of HbGp stability and oxygen carrying capacity with aging, the protein stability is still larger as compared to mammalian hemoglobins. Thus, the extracellular hemoglobins are quite stable and resistant to the auto-oxidation process, making them of interest for biotechnological applications.
•HbGp remains in the reduced and un-dissociated forms for up to two years of storage.•HbGp aging induces the formation of oxidized species and oligomeric dissociation.•HbGp thermal aggregation is promoted by samples aging.•HbGp subunits are the main species for samples with six years of storage.
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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•Acoustic methods inject crystal-containing droplets directly from microplate wells•On-demand acoustic injection uses crystals efficiently without orifices or clogging•Diffraction patterns from crystals measuring several tens of μm are of high quality•Complete datasets can be obtained from fewer than 50,000 crystals
Acoustic droplet ejection provides an automated tool for efficient use of protein crystals in SFX experiments. Roessler et al. used this method to deliver crystal-containing droplets into the XFEL beam to coincide with each X-ray pulse.
In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. ...Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30±4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14±2 ions in the absence of EDTA pre-treatment, and 3±1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn2+ and Ca2+ binding sites. The Ca2+ sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn2+ sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future.
The absence and presence of zinc and calcium ions as promoters of Glossoscolex paulistus hemoglobin protein structural stability: oligomerization, aggregation and enzymatic activity. Display omitted
•We report on the properties of Glossoscolex paulistus hemoglobin (HbGp).•HbGp structure contains around 14 and 30 ions of Zn2+ and Ca2+, respectively.•The ions, Zn2+ and Ca2+, play an important role in maintaining of HbGp structure.•The removal of Zn2+ cations weakens the contacts between the linkers and the dodecamer.•HbGp shows significant antioxidant activity as a peroxidase enzyme with guaiacol.
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples
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