Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Revealing the Dynamic Allosteric Changes Required for Formation of the Cysteine Synthase Complex by Hydrogen-Deuterium Exchange MS.

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design.

Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study.

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

In vitro and in vivo characterization of poractant alfa supplemented with budesonide for safe and effective intratracheal administration.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant.

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.

Surfactant protein C metabolism in human infants and adult patients by stable isotope tracer and mass spectrometry.

Surfactant protein C (SP-C) is deemed as the surfactant protein most specifically expressed in type II alveolar epithelial cells and plays an important role in surfactant function. SP-C turnover in humans and its meaning in the clinical context have never been approached. In this study, we used mass spectrometry to investigate SP-C turnover in humans. We studied four infants and eight adults requiring mechanical ventilation. All patients had no lung disease. Patients received a 24-h continuous infusion of (13)C-leucine as precursor of SP-C, and serial tracheal aspirates and plasma samples were obtained every 6 h till 48 h. SP-C was isolated from tracheal aspirates by sorbent-phase chromatography. (13)C-leucine SP-C enrichment could be successfully measured in three infant and in four adult samples by using mass spectrometry coupled with a gas chromatographer. Median SP-C fractional synthesis rate, secretion time, and peak time were 15.7 (14.1-27.5)%/day, 6.0 (4.7-11.5) h, and 24 (20-27) h. In conclusion, this study shows that it is feasible to accurately determine SP-C turnover in humans by stable isotopes.

Impact of drug administration route on drug delivery and distribution into the lung: an imaging mass spectrometry approach.

During the last decade, significant technological improvements in mass spectrometry have had a great impact on drug discovery. The development of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has set a new frontier for the study of the distribution of endogenous and exogenous molecules present within a tissue. MALDI-IMS is a surface sampling technique that allows not only the detection of multiple analytes but also gives the spatial distribution of those analytes. Active compounds for pulmonary disease need an optimal and well-studied delivery into the lungs, in order to assure distribution with greater penetration into the peripheral or the alveolar region of the lung to maximize the therapeutic effects. IMS is very useful in the field of drug discovery, showing drug delivery and distribution in the body and organs. In this study, we present a comparison between two different ways of carrying out pulmonary drug administration: inhalation of a nebulized aerosol of aqueous drug solutions and intratracheal administration, which is much simpler, not expensive and commonly used during in vivo screening. Tiotropium bromide is a long-acting anticholinergic medicine used for maintenance treatment of chronic obstructive pulmonary disease. In the present work, tiotropium was administered by nebulization and by intratracheal instillation to guinea pigs at doses able to induce significant anti-bronchoconstrictive activity. Lung samples were dissected, frozen, cryosectioned and coated with matrix (α-hydroxy-cinnamic acid). IMS analyses were performed using a MALDI-LTQ-Orbitrap XL. Using this technique we were able to compare different distributions of the drug depending on the method of administration.

Beyond the Interface: Improved Pulmonary Surfactant-Assisted Drug Delivery through Surface-Associated Structures.

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air-liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air-liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: a multidisciplinary characterization of catechol 1,2 dioxygenase.

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Pulmonary Surfactant: A Unique Biomaterial with Life-saving Therapeutic Applications.

Pulmonary surfactant is a complex lipoprotein mixture secreted into the alveolar lumen by type 2 pneumocytes, which is composed by tens of different lipids (approximately 90% of its entire mass) and surfactant proteins (approximately 10% of the mass). It is crucially involved in maintaining lung homeostasis by reducing the values of alveolar liquid surface tension close to zero at end-expiration, thereby avoiding the alveolar collapse, and assembling a chemical and physical barrier against inhaled pathogens. A deficient amount of surfactant or its functional inactivation is directly linked to a wide range of lung pathologies, including the neonatal respiratory distress syndrome. This paper reviews the main biophysical concepts of surfactant activity and its inactivation mechanisms, and describes the past, present and future roles of surfactant replacement therapy, focusing on the exogenous surfactant preparations marketed worldwide and new formulations under development. The closing section describes the pulmonary surfactant in the context of drug delivery. Thanks to its peculiar composition, biocompatibility, and alveolar spreading capability, the surfactant may work not only as a shuttle to the branched anatomy of the lung for other drugs but also as a modulator for their release, leading to innovative therapeutic avenues for the treatment of several respiratory diseases.

Daily Intraperitoneal Administration of Rosiglitazone Does Not Improve Lung Function or Alveolarization in Preterm Rabbits Exposed to Hyperoxia.

Thiazolidinediones (TZDs) are potent PPARγ agonists that have been shown to attenuate alveolar simplification after prolonged hyperoxia in term rodent models of bronchopulmonary dysplasia. However, the pulmonary outcomes of postnatal TZDs have not been investigated in preterm animal models. Here, we first investigated the PPARγ selectivity, epithelial permeability, and lung tissue binding of three types of TZDs in vitro (rosiglitazone (RGZ), pioglitazone, and DRF-2546), followed by an in vivo study in preterm rabbits exposed to hyperoxia (95% oxygen) to investigate the pharmacokinetics and the pulmonary outcomes of daily RGZ administration. In addition, blood lipids and a comparative lung proteomics analysis were also performed on Day 7. All TZDs showed high epithelial permeability through Caco-2 monolayers and high plasma and lung tissue binding; however, RGZ showed the highest affinity for PPARγ. The pharmacokinetic profiling of RGZ (1 mg/kg) revealed an equivalent biodistribution after either intratracheal or intraperitoneal administration, with detectable levels in lungs and plasma after 24 h. However, daily RGZ doses of 1 mg/kg did not improve lung function in preterm rabbits exposed to hyperoxia, and daily 10 mg/kg doses were even associated with a significant lung function worsening, which could be partially explained by the upregulation of lung inflammation and lipid metabolism pathways revealed by the proteomic analysis. Notably, daily postnatal RGZ produced an aberrant modulation of serum lipids, particularly in rabbit pups treated with the 10 mg/kg dose. In conclusion, daily postnatal RGZ did not improve lung function and caused dyslipidemia in preterm rabbits exposed to hyperoxia.

Modulation of expression and polymerization of hemoglobin Polytaur, a potential blood substitute.

Chemically or genetically modified hemoglobins are a therapeutic class indicated for the treatment of a variety of hypo-oxygenation pathologies, severe trauma-related hemorrhages or elective surgery when blood transfusions are refused or not available. Recombinant heterologous hemoglobins offer the possibility of a potentially unlimited production and genetically optimized properties in terms of oxygen affinity, NO reactivity and resistance to autoxidation. Hemoglobin Polytaur is an autopolymerizing human-bovine hybrid mutant, previously obtained as a 500kDa polymer, shown to reduce the infarct volume from focal cerebral ischemia in in vivo animal models. In this work, hemoglobin Polytaur polymerization, carried out under conditions to minimize heme oxidation and modification, resulted in a 180kDa cyclic homogeneous trimer of hemoglobin tetramers. This novel oligomer was characterized by electrophoresis, MALDI-TOF mass spectrometry and gel filtration. The size and the oxygen binding properties were shown to be ideally suited for its use as a blood substitute. Co-expression with the human α hemoglobin-stabilizing protein (AHSP), a chaperone that assists hemoglobin folding in vivo, resulted in an unexpected decrease in yield and in unusual spectroscopic and functional properties, suggesting the formation of strong protein-protein interactions that reduce the expression, hinder the tetramer assembly and prevent purification.

Electrophoretic analysis of PEGylated hemoglobin-based blood substitutes.

Polyethylene glycol (PEG)-conjugated hemoglobins, a novel class of blood substitutes, were investigated by a combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with the microspectrophotometric characterization of single bands and the functional analysis of electrophoretically separated fractions. For these intrinsically heterogeneous products, the molecular mass, the size distribution, and the degree of PEGylation are strictly correlated to their side effects and, therefore, are crucial pieces of information to evaluate their safety and efficacy. The PEGylation pattern was shown to strongly depend on the quaternary conformation of hemoglobin during the reaction, and the degree of conjugation was shown to correlate with the oxygen binding properties of the individual electrophoretically separated fractions. Moreover, small but not negligible fractions of underivatized tetramers, known to be responsible for serious side effects, were detected even in preparations with a high average degree of PEGylation. Overall, this approach might be exploited to characterize other products of protein PEGylation, an increasingly relevant technology for the optimization of the pharmacokinetic properties of protein-based drugs.

Surfactant-Assisted Distal Pulmonary Distribution of Budesonide Revealed by Mass Spectrometry Imaging.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

Absolute quantification of superoxide dismutase (SOD) using species-specific isotope dilution analysis.

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using nondenaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike ((65)Cu, (68)Zn SOD). The stability of the metal-protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 microg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.

PEGylation promotes hemoglobin tetramer dissociation.

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

Evaluation of gel electrophoresis conditions for the separation of metal-tagged proteins with subsequent laser ablation ICP-MS detection.

Although laser ablation (LA)-ICP-MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE-LA-ICP-MS for the separation of metal-binding proteins, focusing on their stability during GE and post-separation gel treatment. The stability of metal-protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal-protein interaction and the principle of separation. We have observed that non-denaturing GE is a suitable separation technique for most metal-protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post-separation treatment of the gel to enable successful detection of the metal. LA-ICP-MS requires drying of the gel without loss of protein-bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA-ICP-MS using silver or Coomassie blue is not recommended, since most protein-bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 microm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.

Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase.