Effect of carbohydrates and polyols on amide content and protein fragmentation in a lactoglobulin preparation

Certain carbohydrates and polyols are used at various stages of the production of immunobiological preparations as stabilizers of biological activity, particularly in the production of lactoglobulin (against opportunistic pathogens) using membrane ultrafiltration. This study concerns the effect of these substances on changes in the amide content in proteins of this lactoglobulin. Lactoglobulin was incubated in near-physiological (0.9% NaCl, pH 5.5) 10% solutions of glucose, fructose, and sorbitol at 4 and 35 degrees C for 7, 14, and 28 days. A lactoglobulin solution in 0.9% NaCl, pH 5.5, was used as the control. All substances studied suppressed the reduction of the amide group content of asparagine and, in contrast, increased the rate of amide group removal from glutamine residues in proteins of lactoglobulin preparations.     

The effect of hydrocarbon addition on the deamidation rate in immune whey proteins

The influence of some hydrocarbons that are often used at different stages of immunobiological preparation's production as stabilizers of biological activity on the dynamics of nonenzymatic deamidation in proteins of immune whey against conditionally pathogenic microorganisms obtained by means of membrane ultrafiltration technology is investigated. Preparations of whey were incubated in 10 per cent solutions of glucose, fructose and sorbitol at the conditions similar to physiological ones (0.9% NaCl, pH 5.5) and temperature of about +4 degrees C and +35 degrees C for 7, 14 and 28 days. A sample dissolved in 0.9% NaCl (pH 5.5) without addition of hydrocarbons was used as a "control preparate". All explored substances brought about the suppressive effect on deamidation rate of asparaginyl residues whereas that of glutaminyl residues, on the contrary, was obviously increased. The possible reasons for these observations are discussed.     

The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus.

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.     

The effect of various chemical compounds and storage conditions on the rate of non-enzymatic deamidation of protein preparations

The effect of different substances partly used as preservatives for the blood storage and at different stages of manufacturing of human blood preparations on the dynamics of nonenzymatic deamidation of commercial protein preparations and on their heat stability was being studied. Albumin and gamma-globulin preparations in the solutions of 60% glycerol, 60% ethylene glycol, 40% beta-alanine, aspartic and glutamic acids in physiological concentrations, 40% glucose and 40% sucrose after 2-hour thermal denaturation (100 degrees) were incubated under (or close to) physiological conditions (pH 7, 37 degrees) for 0.7; 14, 21, 28 and 90 days. The protein preparations in the saline were used as control. After precipitation of protein with TCA, the contents of free ammonia and TCA-soluble products were measured by the Lowry technique. The precipitate washed by organic solvents was used to determine the amide fractions. No reliable difference in increasing the TCA-soluble Folin-positive products was observed. All substances studied but glycerol sped up the deamidation of albumin and gamma-globulin preparations both during thermal denaturation and incubation. On the contrary, glycerol slowed down the deamidation of proteins. Possible reasons of the observed phenomena are discussed.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Improving the analysis of NMR spectra tracking pH‐induced conformational changes: Removing artefacts of the electric field on the NMR chemical shift

pH‐induced chemical shift perturbations (CSPs) can be used to study pH‐dependent conformational transitions in proteins. Recently, an elegant principal component analysis (PCA) algorithm was developed and used to study the pH‐dependent structural transitions in bovine β‐lactoglobulin (βLG) by analyzing its NMR pH‐titration spectra. Here, we augment this analysis method by filtering out changes in the NMR chemical shift that stem from effects that are electrostatic in nature. Specifically, we examine how many CSPs can be explained by purely electrostatic effects arising from titrational events in βLG. The results show that around 20% of the amide nuclei CSPs in βLG originate exclusively from “through‐space” electric field effects. A PCA of NMR data where electric field artefacts have been removed gives a different picture of the pH‐dependent structural transitions in βLG. The method implemented here is well suited to be applied on a whole range of proteins, which experience at least one pH‐dependent conformational change. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.     

Effects of divercin V41 combined to NaCl content, phenol (liquid smoke) concentration and pH on Listeria monocytogenes ScottA growth in BHI broth by an experimental design approach

AIMS: To investigate the main effects and interactions of different factors : divercin V41 (0-4 ng ml(-1)), NaCl content (0.5-5.5% w v(-1)), phenol (liquid smoke) concentration (0-8 ppm), and pH (5.5-7.5) on Listeria monocytogenes ScottA growth. METHODS AND RESULTS: Experiments were carried out in BHI broth using a central composite design. Divercin V41 (div41), NaCl content and pH were found to be the most influential factors whereas phenol concentration in liquid smoke had no effect on L. monocytogenes ScottA growth in our experimental domain. The combined effects of div41, NaCl content and pH decreased L. monocytogenes ScottA maximum specific growth rate (mu(max)) from 0.34 to 0.01 h(-1) and led to a significant increase in lag time (t(lag)) from 5.5 to 25 h. CONCLUSION: In this study, NaCl, pH and phenol conditions were similar to those currently observed in smoked salmon production. This shows that L. monocytogenes ScottA growth could be efficiently delayed by the use of div41 in addition to the usual technological hurdles. SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the technological hurdles of cold smoked salmon production could be further optimized and combined with the use of div41 or the div41 producer strain to improve the food safety of the product.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

Changes in Antioxidative Properties of Lactoferrin from Women's Milk during Deamidation

Spontaneous deamidation of lactoferrin preparations from women's milk was found during incubation for 28 days under simulated physiological conditions (0.85% NaCl, pH 7.0, 37°C). After 28 days of incubation, this deamidation was associated with a 12% decrease in the total amide content in the protein. Addition of deamidated preparation to a suspension of lipoproteins from egg yolk in the presence of Rhodamine 6G decreased the total intensity of rapid and slow emission and also the intensity of the slow emission of Fe²⁺-induced chemiluminescence by 37, 48, and 53%, respectively, suggesting an increase in the antioxidative activity of lactoferrin during deamidation. Deamidation obviously stimulated the nonspecific interaction of lactoferrin with iron ions and, consequently, increased the antioxidant effect of the protein in this way. This was supported by the finding of decreased antioxidative effectiveness of lactoferrin during its complete saturation with iron under the incubation conditions.     

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.     

<Emphasis Type="Italic">Ancylobacter abiegnus</Emphasis> sp. nov., an oligotrophic member of the xylotrophic mycobacterial community

A new species of the genus Ancylobacter, exemplified by strain Z-0056, was isolated from dystrophic humified waters formed by xylotrophic fungi grown on decaying spruce wood. The cells of strain Z-0056 (0.65–0.9 μm) are coccoid, gram-negative, with fimbriae, and nonmotile. The strain is pleomorphic and reproduces by nonuniform division. Strain Z-0056 is an aerobic organoheterotroph and a mesophilic and moderately acidophilic oligotrophic microorganism. As an inhabitant of dystrophic ultrafresh waters, strain Z-0056 is sensitive to NaCl. The bacterium utilizes organic acids (acetate, pyruvate, oxalate, gluconate, malate, succinate, and citrate), as well as xylose and xylan. The microorganism grows in a pH range of 4.0–8.0, with an optimum at pH 5.5. The temperature range for growth is 15–25°C, with an optimum at 20°C. According to its ecophysiological properties, strain Z-0056 belongs to the group of ombrophilic dissipotrophs. The DNA G+C base content is 66.8 mol %. According to phylogenetic analysis, strain Z-0056 belongs to the genus Ancylobacter. Strain Z-0056 showed the highest similarity (98.3%) with the type strain of the species A. oerskovii. The phenotypic and phylogenetic properties of strain Z-0056 support classification of this microorganism within the genus Ancylobacter as the novel species Ancylobacter abiegnus sp. nov.     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Combined effect of salinity and pH on lipid content and fatty acid composition of Tisochrysis lutea

The haptophyte microalga Tisochrysis lutea was heterotrophically grown in F2 medium with different combinations of pH and salinity. Growth, oil content and fatty acids (FAs) profile were determined under each set of conditions. The salinity was adjusted using NaCl at concentrations of 0.4, 0.6, 0.8, or 1.0 M, while pH was adjusted at 7, 8, or 9, and heterotrophic growth was performed using organic carbon in the form of sugar cane industry waste (CM). Fatty acid methyl esters (FAMEs) were identified by gas chromatography. The results showed that pH of 8.0 was the optimal for dry weight and oil production, regardless of the salinity level. At pH 8.0, growth at a salinity of 0.4 M NaCl was optimal for biomass accumulation (1.185 g L^-1). Under these conditions, the maximum growth rate was 0.055 g L^-1 d^-1, with a doubling time of 17.5 h and a degree of multiplication of 2.198. Oil content was maximal (34.87%) when the salinity was 0.4 M and the pH was 9.0. The ratio of saturated to unsaturated FAs was affected by the pH value and salinity, in that unsaturated FAs increased to 58.09% of the total FAs, considerably greater than the value of 40.59% obtained for the control (0.4 M NaCl and pH 8.0).     

Regulation of cell volume and ion concentrations in aHalobacterium

Summary Changes in cell volume and ion content of aHalobacterium species are described in terms of the NaCl concentration (0.5–3.5m) and pH (4–8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O₂ consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.     

Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances

A hydrogen-producing photosynthetic bacteria strain, Rhodopseudomonas acidophila, was used to investigate the production of extracellular polymeric substances (EPS) in the presence of toxic substances and the effect of toxicants on bacterial surface characteristics. Addition of the toxic substances including Cu(II), Cr(VI), Cd(II) and 2,4-dichlorophenol (2,4-DCP) stimulated the production of EPS but reduced the cell dry weight. At concentrations of 30 mg l⁻¹ Cu(II), 40 mg l⁻¹ Cr(VI), 5 mg l⁻¹ Cd(II) and 100 mg l⁻¹ 2,4-DCP, the EPS content increased by 5.5, 2.5, 4.0 and 1.4 times, respectively, than the control. These toxic substances also greatly influenced the proteins/carbohydrates ratio of EPS. The ratios in the presence of toxic substances were always higher than that of control. Furthermore, under toxic conditions, the increase in the protein content far exceeded than that of others in EPS, suggesting that extracellular proteins could protect cells against toxic substances. The toxic substances significantly changed the surface characteristics and flocculation ability of R. acidophila, such as surface energy, relative hydrophobicity and free energy of adhesion.     

pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.     

Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.     

The effect of pH value on the toxicity of culture filtrates ofHelminthosporium Sativum and on its possible correlation to extracellular nitrogen accumulation

Summary Helminthosporium sativum P.K. & B. was grown on modified Fries No. 3 medium buffered at pH 4.7, 5.5, 6.7, and 7.8. The nitrogen content of the culture filtrate, mycelial growth and the toxicity of the filtrate (as measured by its wilt inducing capacity to lupin seedlings) were traced over 24 days. The correlation between nitrogen accumulation and the toxicity of the filtrates was clear in case of media originally buffered at 4.7, 5.5 and 7.8. On the other hand, there was no correlation between growth of the mycelial mats and toxin production. The toxicity of the filtrate was attributed to two substances at least, one of unknown composition produced during the accelerated growth phase, while the other is nitrogenous and produced during the decline growth phase i.e. autolytic in nature. Acidity of the medium favoured toxin production during the first two weeks of incubation. The toxicity of the filtrate remained stable within a wide range of pH between pH 3–9.4.     

pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.