Carbohydrate composition of Carcinus aestuarii hemocyanin.

The hemocyanin of the crab Carcinus aestuarii contains a carbohydrate moiety that represents 1.6% of protein mass. This carbohydrate content is higher than that exhibited by other arthropod hemocyanins so far investigated. By combination of FPLC ion exchange chromatography and reverse-phase HPLC, the native oligomeric protein can be resolved into three major and one minor electrophoretically pure fractions that are found to be homogeneous by N-terminal sequencing and correspond to the subunit polypeptide chains. Sugar analysis on the different subunits reveals that the subunit referred to as Ca2 is glycosylated, with a carbohydrate content of 6.3%. By Ca2 trypsin digestion, separation of glycopeptides, and amino acid sequencing, three consensus sequences for O-glycosylation and one for N-glycosylation were found. MALDI-MS was applied for the determination of the molecular masses of the various glycopeptides and peptides after removal of carbohydrates by neuraminidase and alpha-N-acetylgalactosaminidase.

Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86.

A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.

Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin.

Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.

Fluorescence studies on native and bound to trifluraline soy bean Lb"a" in the enhanced N2 fixation.

The differences in the tryptophan (Trp) fluorescence of native (control) Lb"a" and experimental substance isolated from nodules of the Williams' soy beans variety treated with trifluraline at a concentration of 2.1 x 10(-10) M have been studied. A positively charged environment has been proved for the tryptophans of the native Lb"a" and a negative one for the tryptophans of the experimental Lb"a". The difference in the tryptophan emission spectra at lambdaex = 280 and 300 nm may be assigned to conformational alterations occurring in the experimental Lb"a". This is also confirmed by the greater energy transfer from tyrosine to tryptophan in the experimental Lb"a"--30% compared to the 10% in the native Lb"a". The value of the constant of acrylamide quenching (Ksv = 2.77 M(-1)) shows that the tryptophans are buried more deeply in the experimental Lb"a" than in the native Lb"a" (Ksv = 4 M(-1)). They are substantially lower than Ksv of the standard compound N-Ac-Trp-NH2 (16.30 M(-1)). The activation energy (Ea) of the thermal quenching of tryptophan fluorescence is higher for the experimental Lb"a" (37 kJ mol(-1)) as compared to the standard compound N-Ac-Trp-NH2 (24 kJ mol(-1)) and the native Lb "a" (32 kJ mol(-1)). The dissociation constant of the complex of trifluraline with Lb "a" (6.32 x 10(-11) M) has been determined as well as the stoichiometric ratio trifluraline/Lb"a" (1:1). The estimated nitrogenase activity (microM/gfrw h) and the total Lb (mg/gfrw) for trifluraline are higher as compared to those for the control.

Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits.

The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60 degrees C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, deltaG(D)H2O, toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.

Oxygen transport proteins: III. Structural studies of the scorpion (Buthus sindicus) hemocyanin, partial primary structure of its subunit Bsin1.

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (Mr = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% alpha-helix, 18%, beta-sheet, 19% beta-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.

Spectroscopic properties of a novel neutral proteinase from Saccharomonospora canescens.

A neutral proteinase (NPS) was purified from the culture broth of Saccharomonospora canescens sp. novus, strain 5, using DEAE cellulose and a POROS HQ/M 4.6 x 100 mm column. The stability towards thermal and chemical (guanidine hydrochloride, Gdn.HCl) denaturation of NPS was investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroism and fluorescence spectroscopy. The free energy of stabilization in water was calculated to be 2.1 kcal mol-1. The thermostability was determined by the critical temperature Tc from fluorescence measurements (69 degrees C) and the melting temperature Tm (70 degrees C) from (1) measurements. Quenching with acrylamide, iodide and cesium gives information about the microenvironment of intrinsic protein fluorophores. The Ksv constant for NPS is 4.6 and classifies the emitting tryptophans as 'buried' in the hydrophobic interior of the investigated protein.

Isolation and partial characterization of the N-terminal functional unit of subunit RtH1 from Rapana thomasiana grosse hemocyanin.

Two different structural subunits were identified in Rapana thomasiana hemocyanin: RtH1 and RtH2. RtH1-a is the N-terminal functional unit in the subunit RtH1 and its stability toward temperature and chemical denaturation by guanidinium hydrochloride (Gdn.HCl) are studied and compared with the structural subunit RtH1 and the whole Rapana hemocyanin molecule. The conformational changes, induced by the various treatments, were monitored by CD and fluorescence spectroscopy. The critical temperatures (T(c)) for RtH1-a, the structural subunits and the native Hc, determined by fluorescence spectroscopy, coincide closely with the melting temperatures (T(m)), determined by CD spectroscopy. The free energy of stabilization in water, DeltaG(D)(H(2)O), determined from (Gdn. HCl) denaturation studies, is about two times higher for the structural subunit RtH1 and the whole hemocyanin molecule as compared to the functional unit RtH1-a. The oligomerization between the structural subunits or the eight functional units, assembled in subunit RtH1, has a stabilizing effect on the whole molecule as well as the structural subunits.

A novel Cu,Zn superoxide dismutase from the fungal strain Humicola lutea 110: isolation and physico-chemical characterization.

The fungal strain Humicola lutea 110 produces a mangan- and a copper zinc-containing superoxide dismutases (SOD). In this study, the purification, N-terminal sequence and spectroscopic properties of the new Cu,Zn SOD are described. The preparation of the pure metalloenzyme was achieved via treatment of the strain with acetone followed by gel and ion exchange chromatography. The protein consists of 302 amino acid residues and has a molecular mass of approximately 32 kDa, as determined by PAG electrophoresis and 3100 U mg-1 protein-specific activity. It is a dimeric enzyme with two identical subunits of 15,950 Da, as indicated by SDS-PAGE, mass spectroscopic and amino acid analysis. The N-terminal sequence analysis of the Cu,Zn SOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Conformational stability and reversibility of unfolding of the dimeric enzyme were determined by fluorescence and circular dichroism (CD) spectroscopy. The critical temperature of deviation from linearity (Tc) of the Arrhenius plot ln (Q-1(-1)) vs. 1/T was calculated to be 68 degrees C and the respective activation energy for the thermal deactivation of the excited indole chromophores is 42 kcal mol-1. The melting temperatures (Tm) were determined by CD measurements to be 69 degrees C for the holo- and 61 degrees C for the apo-enzyme. The fluorescence emission of the Cu,Zn SOD is dominated by 'buried' tryptophyl chromophores. Removal of the copper-dioxygen system from the active site caused a 4-fold increase of the fluorescence quantum yield and a 10 nm shift of the emission maximum position towards higher wavelength.

Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110.

A novel thermostable MnSOD was purified to electrophoretic homogeneity from the fungal strain Humicola lutea 110. The preparation of the pure metalloenzyme was performed using treatment with acetone followed by ion exchange and gel permeation chromatography. We found that the activity of this enzyme comprises about 80% of the total superoxide dismutase activity in the crude extract, containing two proteins: MnSOD and Cu/ZnSOD. The MnSOD has a molecular mass of approximately 76 kDa and 7200 U/mg protein specific activity. It is a tetrameric enzyme with four identical subunits of 18 860 Da each as indicated by SDS-PAGE, amino acid analysis and mass spectrometry. N-terminal sequence analysis of MnSOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Physicochemical properties were determined by absorption spectroscopy and circular dichroism measurements. The UV absorption spectrum was typical for an MnSOD enzyme, but displayed an increased absorption in the 280 nm region (epsilon280 = 10.4 mM(-1). cm(-1)), attributed to aromatic amino acid residues. The CD data show that MnSOD has two negative Cotton effects at 208 and 222 nm allowing the calculation of its helical content. The ellipticity at 222 nm is 6800 deg. x m(2) x dmol(-1) and thus similar to the values reported for other MnSODs. The MnSOD from H. lutea 110 is stable over a wide range of pH (4.5-8), even in the presence of EDTA. The enzyme is thermostable at 70-75 degrees C, and more stable than MnSODs from other sources.

Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits.

Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59 degrees C and coincide with the melting temperatures, Tm (49-55 degrees C), determined by CD spectroscopy. The free energy of stabilization in water, delta GDH2O, toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Cal, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.