Characterization of large conformational changes and autoproteolysis in the maturation of a T=4 virus capsid

Nudaurelia capensis ω virus-like particles have been characterized as a 480-Å procapsid and a 410-Å capsid, both with T=4 quasisymmetry. Procapsids transition to capsids when pH is lowered from 7.6 to 5.0. Capsids undergo autoproteolysis at residue 570, generating the 74-residue C-terminal polypeptide that remains with the particle. Here we show that the particle size becomes smaller under conditions between pH 6.8 and 6.0 without activating cleavage and that the particle remains at an intermediate size when the pH is carefully maintained. At pH 5.8, cleavage is very slow, becoming detectable only after 9 h. The optimum pH for cleavage is 5.0 (half-life, ~30 min), with a significant reduction in the cleavage rate at pH values below 5. We also show that lowering the pH is required only to make the virus particles compact and to presumably form the active site for autoproteolysis but not for the chemistry of cleavage. The cleavage reaction proceeds at pH 7.0 after ~10% of the subunits cleave at pH 5.0. Employing the virion crystal structure for reference, we investigated the role of electrostatic repulsion of acidic residues in the pH-dependent large conformational changes. Three mutations of Glu to Gln that formed procapsids showed three different phenotypes on maturation. One, close to the threefold and quasithreefold symmetry axes and far from the cleavage site, did not mature at pH 5, and electron cryomicroscopy reconstruction showed that it was intermediate in size between those of the procapsid and capsid; one near the cleavage site exhibited a wild-type phenotype; and a third, far from the cleavage site, resulted in cleavage of 50% of the subunits after 4 h, suggesting quasiequivalent specificity of the mutation.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Dynamics and Stability in Maturation of a T = 4 Virus

Nudaurelia capensis ω virus is a T = 4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 Å diameter) and have T = 4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP_5.0) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 Å. VLP_5.0 undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent γ peptide (residues 571-641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT_7.6 ↔ NT_5.0). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT_7.6, NT_5.0, and VLP_5.0 at about 8, 6, and 6 Å resolution, respectively. We employed the 2. 8-Å X-ray model of the mature virus, determined at pH 7.0 (XR_7.0), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT_5.0 is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T = 4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic γ peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a γ peptide, while the other is bent with the γ peptide disordered) and dramatic stabilization of the particle. The γ peptide at the flat contact remains dynamic when cleavage cannot occur (NT_5.0) but becomes totally immobilized by noncovalent interactions after cleavage (VLP_5.0).     

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Optimization of bioethanol production during simultaneous saccharification and fermentation in very high-gravity cassava mash

Hydrolysis and fermentation conditions for production of ethanol from very high-gravity cassava mash by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing were optimized using a statistical methodology. During the first part of the study, Placket–Burman design (PBD) was used to study 19 factors that could potentially influence ethanol production. Gravity, particle size, initial pH, and fermentation temperature were identified as key factors that significantly increased final ethanol concentration. The main and interaction effects of these factors were subsequently evaluated based on a quadratic equation generated by central composite design (CCD) using response-surface methodology (RSM). Under the optimized very high-gravity conditions, the final ethanol concentration obtained from experiment increased from 8.21% (wt.%) to 15.03% (wt.%) and was in good agreement with model prediction. By employing two other commercial Saccharomyces strains, similar results were obtained under the same optimized condition. Therefore, we conclude that final ethanol concentration, ethanol productivity (V _P/max), glucose utilization (Y _G/s, Y _P/s), and fermentation efficiency (η _f) were enhanced or maintained under the optimized condition of 40% gravity, 390 μm particle size, initial pH 5.5, and 27°C fermentation temperature.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Backbone resonance assignments for a homolog of the essential ribosome biogenesis factor Fap7 from P. horikoshii in its nucleotide-free and -bound forms

The protein “factor activating Pos9 (Skn7)”, Fap7, is an essential protein in yeast and plays an important role in the biogenesis of the small ribosomal subunit. In eukaryotes, the final processing step of the small ribosomal subunit RNA is the endonucleolytic cleavage of 20S pre-rRNA at cleavage site D yielding mature 18S rRNA. Depletion of Fap7 in yeast leads to a dramatic accumulation of 20S pre-rRNA and a concomitant decrease in 18S rRNA in the cytoplasm. In addition, these cells contain higher levels of 60S, but decreased numbers of 40S ribosomal subunits. Fap7 contains a P-loop like motif placing it in a class with NTPases and kinases and a role for it as an adenylate kinase has been suggested. Up to now both the structure of Fap7 and its detailed function during ribosome biogenesis remain elusive. Here, we present the backbone NMR assignments of a Fap7 homolog from the thermophilic archaeon Pyrococcus horikoshii in its nucleotide free form and bound to the adenylate kinase inhibitor AP₅A.     

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO₂ conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO₂ growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

Toxicity of TiO2 Nanoparticles to Escherichia coli: Effects of Particle Size,Crystal Phase and Water Chemistry

Controversial and inconsistent results on the eco-toxicity of TiO₂ nanoparticles (NPs) are commonly found in recorded studies and more experimental works are therefore warranted to elucidate the nanotoxicity and its underlying precise mechanisms. Toxicities of five types of TiO₂ NPs with different particle sizes (10∼50 nm) and crystal phases were investigated using Escherichia coli as a test organism. The effect of water chemistry on the nanotoxicity was also examined. The antibacterial effects of TiO₂ NPs as revealed by dose-effect experiments decreased with increasing particle size and rutile content of the TiO₂ NPs. More bacteria could survive at higher solution pH (5.0–10.0) and ionic strength (50–200 mg L⁻¹ NaCl) as affected by the anatase TiO₂ NPs. The TiO₂ NPs with anatase crystal structure and smaller particle size produced higher content of intracellular reactive oxygen species and malondialdehyde, in line with their greater antibacterial effect. Transmission electron microscopic observations showed the concentration buildup of the anatase TiO₂ NPs especially those with smaller particle sizes on the cell surfaces, leading to membrane damage and internalization. These research results will shed new light on the understanding of ecological effects of TiO₂ NPs.     

Statistical screening and optimization of process variables for xylanase production utilizing alkali-pretreated rice husk

With the objective of the production of xylanase, local raw material (rice husk) and the indigenous isolate, Aspergillus niger ITCC 7678, were studied. Optimization of the cultivation system for enhancing xylanase production was studied via submerged fermentation. Statistical procedures were employed to study the effect of process variables, such as alkali-pretreated rice husk (as carbon source), NaNO₃ (as nitrogen source), KH₂PO₄, KCl, Tween 80 (as surfactant), MgSO₄, FeSO₄·7H₂O, pH, particle size, agitation, and temperature, on xylanase production by A. niger. The effect and significance of the variables was studied using Plackett–Burman (PBD) and central composite statistical design (CCD). It was found that alkali pretreated rice husk (weight/volume), pH, temperature, and NaNO₃ significantly influence xylanase production. So, these four factors were further optimized by CCD, and it was found that maximum xylanase activity of 10.9 IU/ml was observed at (6.5 % w/v) rice husk, pH (5.5), temperature (32.5 °C), and NaNO₃ (0.35 % w/v) concentration. Under optimum conditions, xylanase production was also studied at the bioreactor level and showed 12.8 % enhanced xylanase activity.     

Preparation and Characterization of Stable pH-Sensitive Vesicles Composed of α-Tocopherol Hemisuccinate

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t_1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.Key words: cleavage, long circulation, PEG-α-tocopherol hemisuccinate, pH-sensitive, vesicles     

Extent and method of grinding of sorghum prior to inclusion in complete pelleted broiler chicken diets affects broiler gut development and performance

A broiler experiment was conducted to examine the effects of sorghum particle size and milling type on the performance, nitrogen corrected apparent metabolisable energy (AME_n), digestive tract development, digesta pH, duodenal digesta particle size and digesta passage rate. Complete pelleted diets with identical botanical and chemical composition containing 750 g/kg whole sorghum (WS), sorghum ground through hammer mill with 1 mm and 3 mm screen (HM1 and HM3) and sorghum ground on a roller mill with 0.15 mm spacing (RM0.15), were made. Sorghum for diets HM3 and RM0.15 were milled to approximately the same mean particle size. Diet WS resulted in poorer (P<0.05) weight gain and feed conversion ratio (FCR) than the other diets from 11 to 21 days of age, while diet RM0.15 resulted in improved FCR. Apparent ME_n determined between 25 and 28 days of age, however, was higher (P<0.05) for diet WS than for the other diets. This was possibly due to a longer adaptation time to a larger feed particle size, as indicated by a lower (P<0.05) pH in the gizzard and smaller duodenal digesta particle size for this diet. Diet HM1 gave similar performance as diet HM3, but resulted in a significantly smaller gizzard, a higher pH of the gizzard content, a lower pH of the duodenal content and larger particles in the duodenal contents, thus indicating that gizzard development and activity were compromised by this diet. Total tract passage rate of the liquid phase marker was slower (P<0.05) in the WS fed birds, but there were no differences in solid phase marker excretion rates.     

Catalytic role of subunit A in ribulose-1,5-diphosphate carboxylase from Chromatium strain D

The oligomeric form of the larger subunit designated as A_m produced by alkali treatment of ribulose-1,5-diphosphate carboxylase from the purple sulfur bacterium, Chromatium strain D, retained partial enzymic activity in the absence of the small subunit (B). Supporting evidence was obtained by polyacrylamide gel electrophoresis at pH 8.9 and Sephadex G-200 gel filtration equilibrated with alkaline buffer at pH 9.2. The specific enzyme activity of A_m (45 nmoles CO₂ fixed/mg protein/min) was approximately 15% of the native intact enzyme molecule. By sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the A_m preparation was proved to be free from contamination of subunit B. With reservation of the sensitivity limit of this particular technique we concur that the larger subunit is the catalytic entity of the carboxylase reaction. The optimum pH of the ribulose-1,5-diphosphate carboxylase reaction catalyzed by isolated A_m lies on the alkaline side at about pH 8.3 with or without Mg²⁺. The undissociated native enzyme possesses an optimum pH on the alkaline side in the absence of Mg²⁺, which shifts to the acidic side in the presence of Mg²⁺. From this behavior it is inferred that the association of the smaller subunit with the larger subunit causes conformational stabilization of the enzyme molecule with an accompanying change in the pH optimum due to Mg²⁺.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Calcium ion binding by tobacco mosaic virus

Calcium ion titrations were performed on solutions of tobacco mosaic virus using a calcium-specific ion-exchange electrode. Scatchard analyses were used to obtain the number of calcium ion binding sites per protein subunit (n) and the apparent stability constant for complex formation (beta' Ca). These experiments were performed on unbuffered solutions, in either water or 0.01 M-KCl, to allow a determination of the number of hydrogen ions released per calcium ion bound (chi). The results indicate that near neutrality, the virus particle possesses two calcium ion binding sites per subunit having apparent stability constants greater than 10(4) M-1. The results are interpreted as if these two sites are non-identical and titrate independently. The higher affinity site for the virus in water has a value of log beta' Ca, which varies from about 8.5 at pH 8.5 to about 3.9 at pH 5.0, and for the virus in 0.01 M-KCl has a value that varies from about 6.2 at pH 8.0 to about 3.7 at pH 5.5. The higher affinity site for the virus in water binds up to two competing hydrogen ions, one with an apparent pKH value greater than 8.5 and the other with a value that varies from 6.0 at pH 5.5 to 7.3 at pH 8.0. For the virus in 0.01 M-KCl, only the competing hydrogen ion binding with an apparent pKH value greater than 8.5 remains. The results could be interpreted as indicating that the electrical charge on the virus particle has a constant value in the pH range 5.5 to 8.0 despite the fact that hydrogen ion titration curves for the intact virus particle indicate that the charge should vary from about -1 per subunit at pH 5.5 to about -4 at pH 8.0.     

Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (αβ)₂ family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 Å containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Å. The functional enzyme resembles previously known α₂ and α₄ endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Factorial design of experiments in the determination of adsorption equilibrium constants for basic methylene blue using biopolymer

Equilibrium conditions in the adsorption of a basic dye on Chitosan were studied. The Factorial Design methods and Analysis of Variance have been applied in the experimental determination of adsorption equilibrium constants. Factorial design with three levels of temperature (30v°C, 45v°C, 60v°C), pH (6.7, 8.1, 9.5), particle size (0.177 mm, 0.914 mm, 1.651 mm) was used in identification of significant effects and interactions in the calculation of the equilibrium constants. The dye adsorption capacity of chitosan was found to increase by decreasing the particle size and increasing temperature and pH. The methodology identifies the principal experimental variables, which have the greatest effect on the adsorption process.