Oligomerization of nitrophorins

Rhodnius prolixus is a blood-sucking insect that uses a mixture of nitrophorin (NP) proteins to deliver nitric oxide (NO) from the insect saliva to the hosts via a ferric heme coordinated to the protein, causing vasodilatation and anticoagulation to support their feeding. R. prolixus NPs 1-4 are very similar proteins ( approximately 20 kDa) with different NO affinities for stepwise NO release triggered by pH increase and histamine binding in hosts. Ultra-high-resolution X-ray structures of native and mutant NPs and their kinetic analysis already have revealed the fundamental steps of NO binding and release. In this study, we found that NPs can exist in multiple oligomerization states at higher concentrations. The oligomers are characterized by a combination of multiple biophysical methods. The intrinsic features of the oligomerization revealed here led us to propose that this intensive, moderately pH- and ligand-dependent oligomerization of NPs has physiological implications in the facilitation of the efficient storage and release of the highly reactive NO in the insect saliva and the victim, respectively.     

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)₃L₃ (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s⁰_20,w values of 58.1 ± 0.2 S and 59.6 ± 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 ± 100 and 3700 ± 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V_bar, for HbGp was performed based on density measurements, providing a value of 0.764 ± 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.     

Interlaboratory comparison study of serum total testoserone measurements performed by mass spectrometry methods

Background

Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays.

Methods

Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis.

Results

The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range.

Conclusions

The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.     

Problems and progresses in speciation of Al in human serum: An overview

Aluminium (Al) is associated with many clinical disorders in renal patients. Al accumulation in brain has also been related to the neurodegenerative processes in Alzheimer’s disease. In order to better understand Al transport in the human body, it is necessary to identify and quantify chemical species in which Al is present in body fluids and tissues. Among a variety of biological samples, Al speciation was the most frequently investigated in human serum. Improvements were made in the development of analytical techniques for the determination of the amount and composition of high molecular mass Al (HMM-Al) and low molecular mass Al (LMM-Al) species in human serum. However, due to the complex chemistry of Al in serum, its low total concentration and the high risk of contamination, speciation of Al in biological samples is still a difficult task for analytical chemists. In this work, problems related to speciation of Al in human serum are critically discussed. An overview of the progress that was made by the use of different analytical procedures, in order to propose analytical protocols for reliable speciation of Al in serum at low ng mL⁻¹ concentration range, is presented.     

Some statistical properties of differencing schemes for baseline correction of sedimentation velocity data

For the detailed analysis of sedimentation velocity data, the consideration of radial-dependent baseline offsets is indispensable. Two main approaches are data differencing (“delta-c” approach) and explicit inclusion of baseline parameters in the model (“direct boundary model” of the raw data). The current work aims to clarify the relationships between the two approaches. To this end, a simple model problem is examined. We show that the explicit consideration of the baseline in the model is equivalent to a differencing scheme where the average value is subtracted from all data points. Pairwise differencing in the delta-c approach always results in higher parameter uncertainty. For equidistant time points, the increase is smallest when the reference points are taken at intervals of 1/3 or 2/3 of the total number of time points. If the difference data are misinterpreted to be statistically independent samples, errors in the calculation of the parameter uncertainties can occur. Contrary to claims in the literature, we observe that there is no distinction in the approaches regarding their “model dependence”; both approaches arise from the integral or differential form of the same model, and both approaches can and should provide explicit estimates of the baseline values in the original data space for optimal discrimination between macromolecular sedimentation models.     

Auto-inhibitory effects of an IQ motif on protein structure and function

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH_664-915 was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH_664-915. Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of I814, Q815, R819, and W827 stretched MYH_664-915 and increased 30-fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin.     

A capillary gas chromatography/mass spectrometric method for the quantification of hydroxysteroids in human plasma

A specific and sensitive methodology for the quantitative determination of hydroxysteroids dehydroepiandrosterone and pregnenolone and their main metabolites in human plasma is described. Hydroxysteroids were extracted using methanol and steroids were further separated by reverse-phase high-performance liquid chromatography, allowing for minimization of the possible chromatographic interferences. Eluted fractions were collected, pooled, and analyzed by gas chromatography-mass spectrometry as trimethylsilyl ether derivatives. The quantification was performed with single-ion monitoring of the highly abundant m/z 129 or m/z 358 fragments. The combination of the chromatographic characteristics to the specific fragments ensured the selectivity and specificity of the method. Under these conditions the method was linear (typical R2 is superior to 0.98 for all hydroxysteroids studied) over the concentration range of 2 x 10(-9) to 10(-6)M with good precision and accuracy.     

Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds' density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D2O/H2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H2O) and 1.1 (D2O) g ml(-1), excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds' method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Delta(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Delta(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein-detergent interactions.     

Design of a microfluidic mixer channel: First steps into creating a fluorescent dye-based biosensor for mAb aggregate detection

A major challenge in the transition to continuous biomanufacturing is the lack of process analytical technology (PAT) tools which are able to collect real-time information on the process and elicit a response to facilitate control. One of the critical quality attributes (CQAs) of interest during monoclonal antibodies production is aggregate formation. The development of a real-time PAT tool to monitor aggregate formation is then crucial to have immediate feedback and process control. Miniaturized sensors placed after each unit operation can be a powerful solution to speed up an analytical measurement due to their characteristic short reaction time. In this work, a micromixer structure capable of mixing two streams is presented, to be employed in the detection of mAb aggregates using fluorescent dyes. Computational fluid dynamics (CFD) simulations were used to compare the mixing performance of a series of the proposed designs. A final design of a zigzag microchannel with 45° angle was reached and this structure was subsequently fabricated and experimentally validated with colour dyes and, later, with a FITC-IgG molecule. The designed zigzag micromixer presents a mixing index of around 90%, obtained in less than 30 seconds. Therefore, a micromixer channel capable of a fast and efficient mixing is hereby demonstrated, to be used as a real-time PAT tool for a fluorescence based detection of protein aggregation.