Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat αγ enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form αα and γγ enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (αα and γγ) of human brain enolase were purified from a crude preparation of the hybrid αγ enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (αα) or a DEAE-Sephadex column (γγ). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified αα and γγ enolases revealed that the two subunits, α and γ, are distinct proteins. The antisera to human αα or γγ enolase cross-reacted with the respective form of rat enolase.     

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells—II

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties.Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic αα form to the neuronal γγ form and contain both enolases as well as the αγ hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both αγ and γγ neuron specific enolases is related to cell maturation and expression of the αγ form precedes that of the γγ form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific αα activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation.As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.     

Properties and application to immunoassay of monoclonal antibodies to neuron-specific γγ enolase

Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations.     

Purification and Properties of γγ-Enolase from Pig Brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as αα- (pI = 6.5), αγ- (pI = 5.6), and γγ-enolase (pI = 5.2). The pI of purified γγ-enolase was also 5.2. The γγ-enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, γγ-enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K _m values for 2-PGA, PEP, and Mg²⁺ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K _m values of other vertebrate enolases. The amino acid composition of pig γγ-enolase, as determined by amino acid analysis, shows strong similarity to the compositions of γγ-enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig γγ-enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig γγ-enolase and the other γγ-enolases are almost identical. Li⁺ proved to be a noncompetitive inhibitor with either 2-PGA or Mg²⁺ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2₁. An R _symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).     

Increased Nervous System-Specific Enolases in Rat Plasma and Cerebrospinal Fluid in Bilirubin Encephalopathy Detected by an Enzyme Immunoassay

Abstract: Three forms of enolase isozymes (αα, αγ, and γγ), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10⁻¹⁸ mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for αα, αγ, and γγ forms, respectively. Levels of αγ and γγ forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of αγ form in blood plasma. These results suggest that assays of NSE in the cerebrospinal fluid or the blood plasma are helpful in detecting neuronal damage in the central nervous system.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Insulin binding to cells of several tissues of the early chick embryo.

The binding of I¹²⁵-labeled insulin to isolated cells from several tissues of the 3- and 4-day chick embryo was determined over a concentration range of insulin from 2 × 10^?11 to 2 × 10^?7M. The cells were obtained from limb bud and nonlimb bud tissues of the 4-day chick, from the headless 3-day chick embryo, and from cartilage of the 12-day embryo. The amount of bound insulin was found to be similar for the cells from the different embryonic tissues. Some implications of these findings for the interpretation of the nature of the binding sites and the teratogenic effect of insulin are discussed.     

Disposition of fucose in brain

Abstract— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium-labelled fucose into brain glyco-proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half-time for the turnover of glycoprotein-fucose in young rats was approximately 2 weeks. Within 3 h after the administration of either [1-³H]fucose or fucose generally labelled with tritium, 75 per cent of the total acid-soluble radioactivity in plasma and brain was found to be volatile, and by 24 h after injection more than 90 per cent of the acid-soluble radioactivity was volatile. The tritium in labelled fiicose appears to undergo arapid exchange reaction with hydrogen atoms in body water, although the tritium in fucose glycosidically- linked to glycoproteins is biologically stable. The rapid disappearance of labelled free fucose from the plasma and tissues of the rat precludes the possibility of any significant degree of reutilization of labelled precursor, and provides support for other data indicating that the turnover of fucose in brain glycoproteins is relatively slow in comparison to that of hexosamine and sialic acid. Activities of α-L-fucosidase in rat brain, with pH optima at 40 and 6.0, had essentially the same K_m (4 × 10^?4 M and 3.2 × 10^?4 M, respectively) with p-nitrophenyl-α-L-fucopyranoside as substrate. Activities of both were competitively inhibited by L-fucose. However, the K_t measured at pH 4 (1.9 × 10^?2) was almost ten times greater than that measured at pH 6 (1.5 × 10^?4).     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.     

A new spectrofluorimetric method for the determination of some tetracyclines based on their interfering effect on resonance fluorescence energy transfer

A rapid, simple, inexpensive and highly sensitive spectrofluorimetric method was developed for the determination of trace amounts of some tetracyclines (TCs), namely tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH) and minocycline hydrochloride (MCH). Binding rhodamine B (RhB) to gold nanoparticles (Au NPs) resulted in quenching of the fluorescence of RhB by a resonance energy transfer (FRET) mechanism, with Au NPs as the energy acceptors. The presence of TCs caused the release of RhB molecules and recovered their fluorescence, and this was used as a basis for the quantitative determination of TCs. The reaction was monitored spectrofluorimetrically by measuring the increase in fluorescence of RhB at 572 nm starting 5 min after mixing the reagents in Tris buffer solution (pH 6.5). The effect of various experimental factors such as buffer type, pH, concentrations of the involved reagents and reaction time were studied to optimize the reaction conditions. Under optimum conditions, the calibration graphs were linear within the ranges 2.08 × 10^?9–1.04 × 10^?6 mol/L, 2.01 × 10^?9–1.00 × 10^?6 mol/L and 2.02 × 10^?9–1.01 × 10^?6 mol/L and detection limits (LODs) of 0.61 × 10^?9, 0.32 × 10^?9 and 0.66 × 10^?9 mol/L were calculated for TCH, OTCH and MCH, respectively, with corresponding percent relative standard deviations (%RSDs) of 1.18, 1.21 and 1.54 (n = 5). The method was successfully applied to the determination of TCs in drinking water, human urine, bovine milk and breast milk samples. Copyright © 2014 John Wiley & Sons, Ltd.     

The migratory history of anadromous and non-anadromous tapertail anchovy Coilia nasus in the Yangtze River Estuary revealed by the otolith Sr:Ca ratio

The migratory history of tapertail anchovy Coilia nasus in the Yangtze River Estuary, China was investigated using otolith Sr:Ca ratios and two-dimensional images of the Sr level from an X-ray electron probe microanalyzer (EPMA). The results showed that 17 of the 22 young-of-the-year (YOY) specimens had low Sr:Ca ratios (1.2–2.4?×?10^?3;1.5?±?0.3?×?10^?3) at the central otolith area, indicating their riverine origin and initial freshwater residence. In addition, 11 of the 14 adult specimens had low Sr:Ca ratios (1.3–2.2?×?10^?3; 1.7?±?0.4?×?10^?3) at the central otolith area but showed alternating changes between high (>4.0?×?10^?3) and low (<2.5?×?10^?3) values outside of this region, reflecting their riverine origin and the migration between freshwater and estuarine habitats. These 28 specimens represented the anadromous population in this region. The other 5 YOY specimens had high Sr:Ca ratios (3.6–5.9?×?10^?3; 4.8?±?0.8?×?10^?3) throughout the life history. Similarly, the other 3 adult specimens had high Sr:Ca ratios (4.0–5.7?×?10^?3; 4.8?±?0.7?×?10^?3) at the central otolith area but showed alternating changes between low and high values outside this region, suggesting that estuarine-origin non-anadromous individuals occurred in this region. The average of the otolith Sr:Ca ratios and Sr level mapping along the life-history transects could be used as a scalar for charting the migratory history of the tapertail anchovy in the Yangtze River Estuary: <2.0?×?10^?3 for freshwater residence and 3.5–6.0?×?10^?3 for estuarine residence.     

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes: II. A comparison of streptonigrin effects on somatic and germ cells

The dose-response relationships for streptonigrin (NSC-45383)-induced chromosome aberrations in rabbit somatic cells are compared with dose-response data derived from the analysis of inherited structural chromosome abnormalities in preimplantation embryos from female rabbits treated with streptonigrin prior to mating. The incidence of inherited aberrations assessed in over 1000 karyotype preparations from 361 6-day blastocysts obtained from 55 female rabbits is used to derive a measure of the transmissible cytogenetic damage induced in the oocytes. The cytogenetic damage assessed in 2300 lymphoblast metaphases from 23 rabbits and 2750 marrow-derived metaphases from 27 rabbits which were collected and prepared for examination 6 h after the initiation of streptonigrin dosing are used to obtain estimates of the somatic cell insult. A uniform maximum likelihood analysis technique is applied individually to the 3 sets of data to derive the coefficients of the dose-response relationships. The resulting equations are Y = 0.6 ± 28.0 (×10^?5) + 8.2 ± 5.1 (×10^?4χ for inherited aberrations in 6-day blastocysts, Y = 9.7 ± 3.3 (×10^?3 + 1.9 ± (×10^?3)χ for bone-marrow cells, and Y = 2.8 ± 0.7 (×10^?2 + 4.8 ± 0.2 (×10^?3)χ for the lymphoblasts. In the somatic tissues Y is the percentage of cells with chromosome breakage, while in the blastocyst data Y is the percentage of 6-day blastocysts with consistent structural chromosome aberrations, and in all equations χ is the total streptonigrin dose in μg/kg.The study shows that streptonigrin injections in the range of 30–90 μg/kg when given to sexually mature female rabbits cause dose-dependent increases in chromosome aberrations in 2 types of somatic cells and in the incidence of inherited aberrations recovered in 6-day blastocysts. The coefficients of damage recovered in blastocysts versus damage recovered in somatic cells have the ratio of 1:2.3:5.8 (blastocysts: bone marrow: lymphoblasts). The results are discussed in terms of risk assessment and kinetics of aberration loss during meiosis and early embryonic development. The conclusion drawn from the study is that somatic cell cytogenetic damage is in some way predictive of damage incurred by oocytes which can be passed on to preimplantation embryos, at least for agents like streptonigrin.     

Tolerance and Immobilization of Cobalt by Some Bacteria from Ferromanganese Crusts of the Afanasiy Nikitin Seamounts

Bacteria isolated from cobalt–enriched ferromanganese crusts on the Afanasiy Nikitin Seamounts in the Equatorial Indian Ocean were examined for their ability to tolerate, and immobilize cobalt in unamended seawater and seawater amended with 0.01% glucose. Retrievable bacterial counts in the form of CFU (colony forming units) on media supplemented with 1 mmol Co l^?1 (58 mg Co l^?1) and 1 mmol Mn l^?1 (54 mg Mn l^?1) were in the range of 1.71 × 10⁴ to 1.05 × 10⁵ gm^?1 (wet wt) of crust, respectively. Most of the isolates (14/24) were pigmented and showed taxonomic affinities to Flavobacterium sp. Two representative isolates were tested for their tolerance of cobalt. We observed that in amended medium, the isolates tolerated up to 1 mmol Co l^?1, whereas in unamended medium they tolerated upto 10 mmol Co l^?1. Microscopic observations of cultures incubated with 10 mmol Co l^?1 showed the occurrence of an extracellular slime layer, which may be responsible for immobilizing the cobalt from the liquid phase. In the unamended medium, the tolerance and stimulation in total cell counts was similar to that in amended medium or sometimes greater. Total cell counts peaked at 100 μmol Co l^?1 for incubations in unamended medium (1.1–2.5 × 10¹¹ cells l^?1) and at 0.1–1 μmol Co l^?1 for incubations in amended medium (1.5–2.6 × 10¹¹ cells l^?1). Counts of formazan-stained respiring cells of both the isolates in the unamended medium reached up to a maximum of 2.9–7.8 × 10¹⁰ l^?1 after incubation for 10 days at 23(±1)°. In the amended medium cell counts of respiring cells attained a maximum in the range of 4.6–15.8 × 10¹⁰ l^?1 at 100 μmol Co l^?1. The Co immobilization rate was on average 82 (± 87.9, n = 24) μmol of Co d^?1. Since the isolates were naturally occurring bacteria from crusts, they could be more environmentally acceptable and safe if used for metal recovery and bio-leaching.     

Protein content and volume of early porcine blastocysts

Mean protein and volume of 222 blastocysts collected on 6 to 9 days of pregnancy were measured. Embryo protein differed (P < 0.05) for each day of development studied. Protein content of embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 μg, respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 μg was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0.56 ± 0.03 × 10^?2mm³ to 1.11 ± 0.04 × 10^?2mm³ between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days of development and females studied. Approximately 20% of all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 of development marks the onset of an exponential increase in embryo protein. Also, blastocyst volume is not correlated with blastocyst protein, suggesting that embryo viability is difficult to estimate by size alone. Further, approximately 20% of the blastocysts collected from a single female may exhibit reduced viability, based on reduced protein content, as early as day 6 of development.     

Fluorescence spectrometric study on the interaction of tamibarotene with bovine serum albumin

The interaction between tamibarotene and bovine serum albumin (BSA) was studied using fluorescence quenching technique and ultraviolet–visible spectrophotometry. The results of experiments showed that tamibarotene could strongly quench the intrinsic fluorescence of BSA by a dynamic quenching mechanism. The apparent binding constant, number of binding site and corresponding thermodynamic parameters at different temperatures were calculated respectively, and the main interaction force between tamibarotene and BSA was proved to be hydrophobic force. Synchronous fluorescence spectra showed that tamibarotene changed the molecular conformation of BSA. When BSA concentration was 1.00 × 10^?6 mol L^?1, the quenched fluorescence ΔF had a good linear relationship with the concentration of tamibarotene in the range 1.00 × 10^?6 to 12.00 × 10^?6 mol L^?1 with the detection limit of 6.52 × 10^?7 mol L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Deoxyribonucleases from rat brain

Two distinctly different DNases were isolated from rat brain and could be separated easily by ammonium sulphate fractionation. One of the DNases acts optimally at pH 5.0 hydrolysing preferentially native DNA and requiring an optimal Mg²⁺ concentration of about 0.03 m . The other DNase has its optimal pH between 7.4 and 8.9, acts preferentially on heat-denatured DNA and requires a lower Mg²⁺ concentration, the optimum being 1 × 10^?4m . Cerebellum from adult rat brain has a lower acid DNase activity and higher alkaline DNase activity, and therefore has a higher ratio of alkaline DNase to acid DNase than the other areas of brain studied. This unique activity ratio in cerebellum of adult rat brain was not observed in infant rat brain.     

Post‐chemiluminescence determination of chloramphenicol based on luminol–potassium periodate system

A post‐chemiluminescence (PCL) phenomenon was observed when chloramphenicol was injected into a mixture of luminol and potassium periodate after the chemiluminescence (CL) reaction of luminol–potassium periodate had finished. The possible reaction mechanism was proposed based on studies of the CL kinetic characteristics, the CL spectra, the fluorescence spectra and the UV‐vis absorption spectra of the related substances. Based on the PCL reaction, a rapid and sensitive method for the determination of chloramphenicol was established. The linear response range was 6.0 × 10^?7–1.0 × 10^?5 mol/L, with a correlation coefficient of 0.9986. The relative standard deviation (RSD) for 5.0 × 10^?6 mol/L chloramphenicol was 2.3% (n = 11). The detection limit was 1.6 × 10^?7 mol/L. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.