Inactivation of poliovirus I (Brunhilde) single particles by chlorine in water

Like the Mahoney strain, the Brunhilde strain of poliovirus aggregated slowly in dilute phosphate-carbonate buffer at pH 6 but not at all at or above pH 7. Infectivity decreased at rates approximately proportional to the concentration of free chlorine present at pH 6 over the entire range of 5 to 40 micrometer. The addition of 0.1 M NaCl to the buffer increased the rate about twofold, but this strain was still twice as resistant as the Mahoney strain. At pH 10, inactivation was much slower than at pH 6, but when 0.1 M NaCl was added, the rate was increased 31-fold, making the OCl- at pH 10 over three times more effective than HOCl at pH 6.     

Inactivation of coxsackieviruses B3 and B5 in water by chlorine

The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6.     

Inactivation of coxsackieviruses B3 and B5 in water by chlorine.

The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6.     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.     

Surface areas of 1-palmitoyl phosphatidylcholines and their interactions with cholesterol.

The activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow linear decrease in velocity as the concentration of NaCl was increased. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. The order of efficiency of added glucosyl acceptors in beta-glucosidase-catalysed reactions was found to be fructose greater than sucrose greater than glycerol greater than methanol. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated and an intimate glucosyl cation-carboxylate ion-pair intermediate and an alpha-glucosyl enzyme intermediate [Umezurike, G. M. (1981) Biochem. J. 199, 203-209]. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of beta-glucosidase in the presence of added glucosyl acceptors or of NaCl.     

The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.     

Process optimisation for anion exchange monolithic chromatography of 4.2 kbp plasmid vaccine (pcDNA3F)

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30–80 °C), mobile phase flow rate (0.1–1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50–80%), buffer pH (6–10), ionic strength of binding buffer (0.3–0.7 M) and buffer gradient elution slope (1–10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400 nm pore size of monolith in 0.7 M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0 M at 3%B/min.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

A light-scattering study of the effect of calcium chloride on the molecular weight of Busycon hemocyanin.

Solutions of Busycon canaliculatum have been studied by light scattering. In 0.05 M Trizma buffer +0.1 M NaCl at pH 7.0 at 14 degrees, the weight-average molecular weight is 8.9 X 10(6). In the presence of added CaCl2 (0.02 M), the molecular weight of the protein increases to 10.7 X 10(6), and the second virial coefficient is reduced. At pH 9.95, the molecular weights with and without 0.02 M CaCl2, are 3.7 X 10(6) and 1.3 X 10(6), respectively; and the effect of Ca++ in reducing the second virial coefficient is much greater than at pH 7.0. These results can be understood on the basis that at pH 7.0, ca++ increases the association of hemocyanin, by binding and intermolecular linkage through the carboxyl groups of protein side chains. At pH 9.95, amino groups are deprotonated and therefore also become available for Ca++ binding. The relative effect of Ca++ in enhancing the association of hemocyanin therefore becomes greater at the higher pH.     

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.     

Partial characterization of thymocyte-activating factor derived from MDP-stimulated guinea pig fibroblasts

The activity of fibroblast-derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE-Sepharose CL-6B in Tris-HCl buffer at pH 8.0, and was eluted with 0.1-0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL-6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70-110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T-cell-activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.     

Some Properties of the Autolytic N-Acetylmuramidase of Lactobacillus acidophilus

The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 mug/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Collagenase Digestion for Distinguishing Neural from Reticular Fibres in Silver Stains

Fresh frozen sections of liver and duck salt gland, 20 μ thick were attached to slides and immersed for 20 min in Carnoy's 6:3:1 fixative; washed 3 times with 0.9% NaCl; placed in M/15 phosphate buffer, pH 7.0 for 20 min; then incubated for 5 hr at 37 C in a 0.1% solution of collagenase (Koch-Light Laboratories) in the phosphate buffer. After washing in 0.9% NaCl the slides were immersed for at least 24 hr in 4% formaldehyde (10% formol-saline). Slides were examined for morphological detail after haematoxylin and eosin staining, for nerve fibres after silver impregnation, and for connective tissue fibres. Attempts to use papain and pepsin digestion on sections after similar fixation were not successful, as much of the tissue was destroyed.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect

Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.