Spectroscopy - Volume 18, issue 2

Authors: Godderz, LeAnn J. | Rodgers, Karla K.

Article Type: Research Article

Abstract: The recombination activating gene products (RAG1 and 2) catalyze the initial DNA cleavage steps during V(D)J recombination for the diversification of the immune repertoire. As the fundamental properties of the RAG proteins remain largely unknown, our objective is to investigate the self‐association and conformational properties of RAG1. To analyze RAG1 association and dissociation, a time course of multi‐angle laser light scattering measurements (MALL SEC) was performed on samples at different oligomerization states over a wide range of ionic strengths. The molecular masses of the predominant RAG1 species corresponded to dimer, tetramer, and a previously uncharacterized octamer state. Furthermore, the fraction …of RAG1 in the tetrameric and octameric states increased significantly over time at lower ionic strengths, indicating that these oligomeric forms may factor into the physiological function of RAG1. Circular dichroism (CD) analysis of RAG1 showed a significant dependence in secondary structure on ionic strength with changes in α‐helical content over time that may correspond to the changes in oligomerization states shown by MALLS SEC. Together, the MALLS SEC and CD analyses of RAG1 self‐association properties and secondary structure give further insight into formation of the protein complex responsible for catalyzing V(D)J recombination. Show more

Citation: Spectroscopy, vol. 18, no. 2, pp. 311-322, 2004

Authors: Kopecký, Vladimír | Ettrich, Rüdiger | Hofbauerová, Kateřina | Baumruk, Vladimír

Article Type: Research Article

Abstract: This work introduces a new approach connecting vibrational spectroscopy with homology and energetic molecular modeling of proteins. Combination of both methods can compensate their disadvantages and result in realistic three‐dimensional protein models. The approach is most powerful for membrane proteins or glycoproteins with high carbohydrate content where X‐ray or NMR analysis is not always successful. Nevertheless, it can also serve as a tool of preliminary analysis of any protein with unknown structure. Power of the approach is demonstrated on human α1 ‐acid glycoprotein. Its predicted structure published in [V. Kopecký Jr. et al., Biochem. Biophys. Res. Commun. 300 (2003), 41–46] …is discussed in detail with respect to the approach and its general employment. Show more

Keywords: Raman spectroscopy, Fourier transform infrared spectroscopy, molecular modeling, orosomucoid, binding site, thermal dynamics

Citation: Spectroscopy, vol. 18, no. 2, pp. 323-330, 2004

Authors: Maccarrone, Mauro | Di Venere, Almerinda | van Zadelhoff, Guus | Mei, Giampiero | Veldink, Gerrit | Rosato, Nicola | Finazzi‐Agrò, Alessandro

Article Type: Research Article

Abstract: Lipoxygenases (Loxs) form a homologous family of non‐heme, non‐sulfur iron containing lipid‐peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase‐1 (Lox‐1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox‐1 is known to produce a 60 kDa fragment, termed “mini‐Lox”, which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi‐Agrò, Biochemistry 40 (2001), 6819–6827). In this study, we have investigated the …stability of mini‐Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini‐Lox is a rather unstable molecule, which undergoes a two‐step unfolding transition. Both chemical‐ and physical‐induced denaturation suggest that mini‐Lox is more hydrated than Lox‐1, an observation also confirmed by 1‐8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate‐induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini‐Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox‐1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini‐Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency. Show more

Keywords: Limited proteolysis, intrinsic fluorescence, conformational changes, ANS binding

Citation: Spectroscopy, vol. 18, no. 2, pp. 331-338, 2004

Authors: Mendes, M.A. | Souza, B.M. | Marques, M.R. | Palma, M.S.

Article Type: Research Article

Abstract: The effect of salts, detergents and chaotropic agents on mass spectrometric analysis are relatively well understood, mainly due to their actions decreasing the performance of ESI interface in mass spectrometric analysis. However, there are few studies in the literature characterizing the effect of protein stabilization by glycerol, followed in some circumstances by the suppression of protein signal when ESI interface is used. The aim of the present research was to investigate in details the mass spectrometric behavior of some proteins in presence of high levels of glycerol during ESI–MS analysis. Thus, horse heart myoglobin and chicken ovalbumin were used as …standard proteins. It was demonstrated that the presence of 1% (v/v) glycerol suppressed the signal of these proteins during the ESI–MS analysis, even when the sample nozzle potential was scanned from 28 to 80 V. However, when the glycerol concentration was decreased to 0.5% (v/v) and the sample cone voltage adjusted to 50 V, a perfect envelope of peaks was observed, allowing the spectrum deconvolution and the molecular mass determination with mass accuracy lower than 0.01% in each situation. A molecular explanation for this suppressive effect and for the analytical overcoming of this difficult is proposed. Show more

Citation: Spectroscopy, vol. 18, no. 2, pp. 339-345, 2004

Authors: König, Simone | Wagner, Frank | Kauschke, Ellen | Eue, Ines

Article Type: Research Article

Abstract: Annelids are capable of defending themselves from pathogens and of recognizing degenerated self tissue. These reactions require specialized immune mechanisms that are effected by proteins and cellular reactions. Hemolytic proteins are the most striking humoral defense molecules in the earthworm Eisenia fetida. Beside their hemolytic activity these proteins possess agglutinating, antibacterial, cytotoxic, and clotting properties. Hemolytic proteins both from coelomocytes (CL39,41 ) and coelomic fluid (H1−3 ) of wildtype E. fetida were isolated and assigned to fetidin and lysenin using mass spectrometry and bioinformatic tools. Glycosylation was found for H1 . In silico analyses of the hemolysins revealed two hemolysin …isoforms and consensus sites for N‐glycosylation and peroxidases proximal heme‐binding ligand. Show more

Keywords: Eisenia fetida, hemolysins, mass spectrometry, isoforms, glycosylation, hydrogen peroxide

Citation: Spectroscopy, vol. 18, no. 2, pp. 347-353, 2004

Authors: Hamilton, Brett | Díaz Sierra, Mónica | Lehane, Mary | Furey, Ambrose | James, Kevin J.

Article Type: Research Article

Abstract: The azaspiracids, AZA1, AZA2 and AZA3, are the predominant shellfish toxins responsible for the human toxic syndrome, azaspiracid poisoning. Collision induced dissociation (CID) mass spectra were generated for azaspiracids using nano‐electrospray ionisation (ESI) with a hybrid quadrupole time‐of‐flight (QqTOF) mass spectrometer in positive mode. Six main backbone fragmentations of the polyether skeleton of azaspiracids were observed as well as multiple neutral losses of water molecules from the parent and product ions. The characteristic charge‐remote fragmentation of the carbon skeleton of azaspiracids produced nitrogenous ions. The three azaspiracids differ from one another by 14 Da due to methylation in the A‐ …and E‐rings. Three fragmentation pathways, involving cleavage of the E‐ring, C27–C28 and G‐ring, gave ions that were common to all azaspiracids. Another three fragmentations involving the A‐ring, C‐ring and C19–C20, were useful for distinguishing between azaspiracid analogues. Multiple tandem ion‐trap mass spectrometry (MSn ) was used to confirm the fragmentation pathways. Show more

Citation: Spectroscopy, vol. 18, no. 2, pp. 355-362, 2004

Authors: Schilling, Stephan | Demuth, Hans‐Ulrich

Article Type: Research Article

Abstract: Glutaminyl cyclase (QC, EC 2.3.2.5) catalyses the formation of pyroglutamyl residues from glutamine at the N‐terminus of peptides and proteins. In previously applied assays, QC activity was determined by either analysing the products formed using HPLC coupled with photometric or fluorometric detection, radioimmunoassay, or by detecting the release of ammonia spectrophotometrically. Although these methods are sensitive, they are all discontinuous and therefore time‐consuming and laborious. To conduct a detailed kinetic investigation of QC catalysis, we developed coupled continuous assays suitable for microplates which allow now convenient determination of QC activity. The methods either use pyroglutamyl aminopeptidase or glutamate dehydrogenase as …auxiliary enzymes, which results in the liberation of chromophores or fluorophores such as pNA, AMC, βNA or in the conversion of the chromophore NADH/H+ into NAD+ , respectively. The assays were applied in various enzyme isolation and characterisation studies, using crude protein solutions as well as purified enzyme in pH‐dependence, substrate and inhibitor specificity investigations. Depending on the respective analytical task, both assays complement each other. Therefore, different enzymatic properties could be explored in more detail. Since the employed strategy of assay development could be of interest also for the analysis of other enzymes, the methods are described here in a comprehensive manner. Show more

Citation: Spectroscopy, vol. 18, no. 2, pp. 363-373, 2004

Authors: Grassi, Mario | Voinovich, Dario | Grabnar, Iztok | Franceschinis, Erica | Perissutti, Beatrice | Filipovic‐Grcic, Jelena

Article Type: Research Article

Abstract: The potential of a sustained release formulation for paracetamol produced by melt pelletisation was investigated. After the production of the pellets, based on the combination of stearic acid as a melting binder and anhydrous lactose as a filler, the 3000–2000 μm size fraction was selected in the light of the promising in vitro dissolution results for further characterisations, including scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS), specific surface area and true density determination. Hence the release mechanism was analysed with the help of an appropriate mathematical model. The mathematical model was built on the hypotheses that drug diffusion and …solid drug dissolution in the release environment are the key phenomena affecting drug release kinetics. Bioavailability of the developed formulation was evaluated in an in vivo study in eight subjects. Show more

Keywords: Melt pelletisation, high shear mixer, paracetamol, sustained release, in vitro dissolution, modelling of drug‐release mechanism, in vivo bioavailability studies

Citation: Spectroscopy, vol. 18, no. 2, pp. 375-386, 2004

Authors: Scott, Jill R. | Ham, Jason E. | Durham, Bill | Tremblay, Paul L.

Article Type: Research Article

Abstract: Metal polypyridines are excellent candidates for gas‐phase optical experiments where their intrinsic properties can be studied without complications due to the presence of solvent. The fluorescence lifetimes of [Ru(bpy)3 ]1+ trapped in an optical detection cell within a Fourier transform mass spectrometer were obtained using matrix‐assisted laser desorption/ionization to generate the ions with either 2,5‐dihydroxybenzoic acid (DHB) or sinapinic acid (SA) as matrix. All transients acquired, whether using DHB or SA for ion generation, were best described as approximately exponential decays. The rate constant for transients derived using DHB as matrix was 4×107 s−1 , while the rate …constant using SA was 1×107 s−1 . Some suggestions of multiple exponential decay were evident although limited by the quality of the signals. Photodissociation experiments revealed that [Ru(bpy)3 ]1+ generated using DHB can decompose to [Ru(bpy)2 ]1+ , whereas ions generated using SA showed no decomposition. Comparison of the mass spectra with the fluorescence lifetimes illustrates the promise of incorporating optical detection with trapped ion mass spectrometry techniques. Show more

Citation: Spectroscopy, vol. 18, no. 2, pp. 387-396, 2004