Purification and structural characterization of recombinant rat gamma-interferon from Escherichia coli

An efficient method has been developed for the purification of recombinant rat gamma-interferon (rat rIFN-gamma). The procedure involves extraction of the Escherichia coli cell paste with 6 M guanidine-HCl (GuHCl), adsorption of the rat rIFN-gamma onto C8 alkyl-bonded silica, and elution with 50% propanol. The protein is essentially pure at this step, but is quantitatively precipitated by threefold dilution with aqueous buffer at pH 8.5. The precipitate is then dissolved with 6 M GuHCl in a buffer containing 0.05%. Tween-80 to about 0.3 mg/ml and dialyzed against the same buffer. The rat rIFN-gamma, which remains soluble on dialysis is again precipitated by dialysis against ammonium sulfate at 80% saturation. This final precipitate is readily soluble in 0.1 M ammonium acetate buffer, pH 8.5. The preparation is fully active and possesses a specific activity of 2-6 X 10(6) units/mg. The recoveries ranged from 50 to 85% in several experiments. The sequence of 20 amino acid residues from the NH2-terminus of the protein was determined using an automated sequencer and was found to agree with that deduced from the cDNA sequence.     

Preparation of giant liposomes

A method is described for the preparation of giant unilamellar lipid vesicles that are stable in electrolyte solution. In general, it involves dialysis of lipid and indifferent solute in a water-miscible organic solvent against an aqueous buffer. During dialysis the concentration of organic solvent decreases so that vesicles form under conditions where their internal contents are continuously hyperosmotic. Interlamellar attractive forces are neutralized, even between bilayer membranes with no net charge, and giant vesicles are generated in large numbers. The population is heterogeneous but most large vesicles have diameters between 10 and 100 μm. The method is simple. One procedure involves dialysis for a day or more of a methanol solution of phosphatidylcholine, supersaturated with methylglucoside, against an aqueous phase containing up to 1 M univalent electrolyte. The procedure is effective over a wide range of temperature and pH.     

Validated high performance liquid chromatography-UV detection method for the determination of daptomycin in human plasma

Daptomycin is the first approved member of the new class cyclic lipopeptide antibiotic drugs, effective against a broad spectrum of Gram-positive bacteria. Here we present an HPLC method with UV detection capable to obtain pharmacokinetic data of daptomycin in human plasma, exemplarily shown in a critically ill patient with acute renal failure undergoing extended daily dialysis. Sample preparation consists only of protein precipitation with methanol. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C8 column and daptomycin was detected at 224 nm. The calibration function was linear over the range from 3.5 to 350 microg/ml. The relative standard deviations were <2% in the intra-day and <6% in the inter-day measurements. The accuracy was always better than 7%. Daptomycin was stable in aqueous solutions for 2 months frozen at-20 degrees C. However, in plasma frozen at -20 degrees C a loss of 25% in 1 month was observed.     

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.     

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.     

Prospects of designing medicines with diverse therapeutic activity on the basis of dinitrosyl iron complexes with thiol-containing ligands

A stable hypotensive preparation (Oxacom) based on dinitrosyl iron complexes (DNIC) with glutathione has been developed. The preparation has successfully passed pharmacological trials. Tests on volunteers have shown a high hypotensive activity of the preparation: a single intravenous infusion of its aqueous solution at a dose of 0.2 μmol active substance per kg body wt led to a 20–30% decrease in arterial pressure, which persisted for 15–20 h. Similar experiments on animals demonstrated that aqueous solutions of DNIC with cysteine or glutathione also exert a hypotensive action due to their vasodilatory activity. Besides, these complexes accelerate wound healing and produce a potent erectile effect. There is reason to suppose that DNIC with thiol ligands as NO donors may be cytotoxic for pathogenic mycobacteria Mycobacterium tuberculosis and, after appropriate treatment, inhibit cancer cell proliferation. These complexes can be used as analgesics, for inhibiting the adhesion process, in treating preeclampsia, spermatogenesis pathologies, and in cosmetology for treatment of skin injury.     

A Nuclear Stain for Escherichia Coli And Related Organisms

The dye base of new fuchsin was precipitated by adding potassium hydroxide to the dye solution. The precipitate was filtered out and washed with water. It was then suspended in water, brought into solution and adjusted to a pH of about 5.0 with nitric acid. The staining solution was prepared by adding 0.3 ml. of a 14% aqueous solution of pyrogallol and 0.1 ml. of a 1% aqueous solution of boric acid to 3.0 ml. of the dye solution. Smears of cells were made in water on a slide and allowed to dry before covering with the staining solution which was also permitted to air dry. The smear was then washed in water and mordanted for 5-20 seconds in a 0.1% aqueous solution of mercuric nitrate. After rinsing in water, the smear was air dried. When dry, the slide was placed on a 50° C. warm plate for a few seconds before covering with a very thin film of a 5% aqueous solution of nigrosin which had a pH of about 5.0.     

Study of the association of nystatin and amphotericin B in nonaqueous solvent systems]

Association of nystatin and amphotericin B in non-aqueous systems was studied with the method of equilibria dialysis. A specially treated celophane membrane arresting colloid associats and macromolecules with a molecular weight of more than 30000 in the systems of dimethylformamide-ethylacetate was used for the dialysis. Relation between the dialysis rate and the difference of the concentrations at every side of the membrane was used for estimation of the antibiotic colloid association level. It was found that nystatin formed stable associates of the colloid type in the system of dimethylformamide-ethylacetate, close by the composition to the critical one or that providing precipitation of the antibiotic. Unlike nystatin, amphotericin B formed not colloid but larger conglomerates which precipitated. Neither of the antibiotics formed colloid associates in dimethylformamide. The level of the nystatin colloid association increased with a rise in the solution concentration and reached 80%. On the basis of the results obtained the following supposition concerning the mechanism of formation of the antibiotic complexes with polyvinylpyrrolidone (PVP) in non-aqueous systems is possible: sorption of PVP on the colloids formed or larger associates of the polyenic antibiotics must take place during coprecipitation which is accompanied by formation of a precipitate of the sorption complex.     

Effects of angiotensin II and of phorbol ester on protein kinase C activity and on prostacyclin production in cultured rat aortic smooth-muscle cells.

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

Analysis of an Effective Antibiotic (Chaetomacin) Isolated from a Thermophilic Bacillus sp. Against Olive Green Mold

Successful methods to control the damaging weed mold Chaetomium olivaceum (olive green mold) in mushroom beds are not known. An effective antibiotic (named chaetomacin) against C. olivaceum was isolated from a thermophilic Bacillus sp. This compound was shown to be an extremely potent and stable antibiotic, effective over a wide range of both pH (2 to 10) and temperature (−15 to 150°C). Chaetomacin is soluble in most polar solvents and insoluble in nonpolar solvents. It is produced only at mesophilic temperatures and is also active against other Bacillus spp. and various eucaryotes, but it demonstrates no activity against gram-negative rods or gram-positive cocci. Final purification of chaetomacin was accomplished through thin-layer chromatography on silica gel analytical plates. Amino acid analysis revealed the antibiotic to be a peptide, acidic in nature. Examination of the literature reveals no other previously isolated antibiotics identical to chaetomacin.     

Study of oxytetracycline solubility in an aqueous medium]

Solubility of oxytetracycline dihydrate in aqueous media was studied. It was shown that solubility of the drug in bidistillate at a temperature of 20 degrees was 195 gamma/ml, which was much lower than the requirements of some pharmacopoeia with respect to the drug solubility. Dependence of oxytetracycline dihydrate solubility in the aqueous medium on the values of pH and temperature was found. Indigency of the procedure described in the literature for determination of antibiotic solubility according to the dry weight of the filtrate on addition of large excesses of the solid phase to the system was shown.     

Preparation and Properties of Nanocolloidal Rhenium Sulfide Solution for Lymphoscintigraphic Methods of Micrometastase Examination

A method for the preparation of a nanocolloidal solution of rhenium sulfide is proposed. It includes the following stages: interaction of ammonium perrhenate and sodium thiosulfate in an aqueous solution of gelatin in an acidic medium with heating to 70–80°C; neutralization to pH 7.0; dialysis against saline; desalination and concentrating by ultrafiltration. The resulting colloidal solution contains 85% of particles with an optimal size of 80–100 nm and can be used as a diagnostic tool for lymphoscintigraphy of sentinel lymph nodes in patients with cancer and other diseases.     

Inhibition of rhizobia by a strain of Rhizobium trifolii: Some properties of the antibiotic and of the strain

Summary An ineffective strain of Rhizobium trifolii, T24, produced an antibiotic active against other strains from several species of Rhizobium. The antibiotic was relatively stable to heat treatment below 90° C but was irreversibly inactivated by low pH and by unidentified microbial contaminants. Activity was readily destroyed by papain but not by trypsin, chymotrypsin, lysozyme, -amylase, ribonuclease or deoxyribonuclease. The molecular weight of the active substance was estimated by gel filtration and dialysis procedures to be within the range of 1000 to 2000, probably at the lower end of this range. Growth inhibition of sensitive rhizobia appeared to occur mainly by bacteriostasis, and inhibition was reversible by some basic metabolites. Effective nodulation of clover seedlings by antibiotic-sensitive strains of R. trifolii was strongly suppressed by mixed inocula including T24. Predominance of ineffective symbiosis was apparently due to competitive advantage associated with antibiosis.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

The Semi Large-scale Production, Extraction, Purification and Properties of an Antibiotic Produced by Pseudomonas fluorescens Strain 175

S ummary : Production, extraction, purification and properties of an antibiotic produced by Pseudomonas fluorescens , strain 175, are described. Extraction efficiency was c. 57% with 3–5 g of product/350 1 batch culture, the product having an activity of 7900 units/mg (0· 12 μg/ml) against Staphylococcus aureus Paler. The antibiotic had the properties, of a weak acid and was active against both Gram positive and Gram negative bacteria; it was stable over a wide pH range, was bactericidal, inactivated by serum, had low toxicity and was haemolytic at relatively high concentrations.     

Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [¹⁴C]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (0–37°C). Moreover, owing to the flexibility in the reaction conditions afferded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attalned.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.