phenyl-Sepharose Column Chromatography Research Articles

Protein Complex of Fibrinogen Gamma Chain and Complement Factor H in Ovarian Cancer Patient Plasma.

In the plasma of an advanced cancer patient, fibrinogen is sometimes increased with possible effects on red blood cells (RBCs). The plasma fraction deteriorating osmotic resistance of RBCs was separated from a patient's plasma with advanced ovarian cancer by phenyl-sepharose column chromatography and analyzed with gel filtration chromatography. In the plasma fraction, we found a protein reactive against whole fibrinogen with a molecular weight higher than that of intact fibrinogen from a healthy volunteer. The-high molecular weight protein was immunoractive to an antibody against fibrinogen gamma chain but not to an antibody against alpha or beta chain. Complement factor H, identified by N-terminal sequencing of a 150-kDa protein separated from the protein, was also eluted from anti-fibrinogen gamma immunoaffinity column. Fibrinogen gamma chain and complement factor H were found to be bound as a protein complex in the plasma of a patient with advanced ovarian cancer.

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium, Salinicoccus iranensis strain QW6.

Oxyanions of tellurium, like tellurate (TeO4 (2-)) and tellurite (TeO3 (2-)), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO3 (2-) resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.

Production and Characterization of Extracellular phytase: An Industrial Enzyme.

Microbial enzymes meet industrial demands. Over 60% of the total Phosphorus in cereal grains and oil seeds as well as their by-products is found as phytate, myo-inositol hexakisphosphate. Phytases are a group of enzymes that initiate the phosphate hydrolysis from phytate and produce myo-inositol and inorganic phosphate by catalyzing the stepwise removal of the phosphate groups. Microbial phytases effectively improve dietary phytate-P utilization. Extracellular phytase produced by Bacillus subtilis ATCC 6051 was purified by acetone precipitation, DEAE-sepharose and phenylsepharose column chromatographies. The molecular weight of the enzyme was estimated to be 48 kDa on gel filtration and 43kDa on SDS-polyacrylamide gel electrophoresis. Its optimum pH and temperature for phytase activity were pH 6.2 to 8.2 and 40°C without 10mM CaCl2 and pH 6.5 to 9.5 and 60° with 10mM CaCl2. The enzyme activity was stable from pH 6.5 to 10. The enzyme had an isoelectric point of 6.8. It was very specific for sodium phytate. The Km value for sodium phytate was 50μM. Its activity was inhibited by EDTA and metal ions such as Mn2+, Hg2+, Cu2+, Cr3+, Co2+, Ba2+ and Cd2+ ions. The enzyme has great potential in food industry, probiotics, animal feed supplement and transgenic crops.

A chitosanase purified from the snail of Achatina fulica

A chitosanase from the snail of Achatina fulica was purified 18.27-fold with 0.68% recovery of protein and 12.53% recovery of enzymatic activity by phenyl-sepharose column chromatography, diethylaminoethanol (DEAE)-sepharose column chromatography and Sephacryl S-300 gel filtration. The molecular mass of chitosanase was 72 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the isoelectric point (pI) of purified chitosanase was 5.45 estimated by isoeletrofocusing electrophoresis. The Km measured for the chitosanase was 0.154 μM, with an apparent V max of 0.005 μM/min. The current work for the first time obtains a chitosanase from A. fulica might be potentially served as a useful enzyme for hydrolyzing chitosan. It is purified as an important initial material for mass spectrometric sequencing, gene cloning and protein expression of this chitosanase. Keywords: Achatina fulica , chitosanase, purification, characterization

Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563.

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS–PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5–9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5–9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag+, Hg2+ (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The K m for Phy I and II for sodium phytate was 2.01 and 0.145 mM while V max was 5,018 and 1,671 μmol min−1 mg−1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.

Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization

An endochitinase was previously purified and the gene was cloned from the psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc)2 was used as a substrate, the specific activity of the enzyme was determined to be 20U/mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)2 are used as substrates. Optimal activity for rChi21702 was observed at 37°C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10°C and 44% activity at 0°C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.

Characterization of a 27 kDa Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CMSephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and 45 degrees , respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E.colo-Bacillus Shuttle vector, pHY300PLK.

Inhibition of Human Dimethylarginine Dimethylaminohydrolase-1 by S-Nitroso-L-homocysteine and Hydrogen Peroxide

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys(274) and His(173) are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (0.088 M(-1) s(-1)), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M(-1) s(-1)). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.

Cloning and Expression of Cyclodextrin Glycosyltransferase Gene from Paenibacillus sp. T16 Isolated from Hot Spring Soil in Northern Thailand

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Staphylococcus epidermidis urease의 정제 및 생화학적 특성에 관한 연구

본 연구에서는 피부상재균이며 기회병원균이기도 한 Staphylococcus epidermidis ATCC12228로부터 urease효소를 4단계 크로마토그라피 방법을 사용하여 1,127배 정제하고 그 생화학적인 특성을 규명하였다. 정제된 urease 효소는 SDS-PACE 전기영동분석 및 gel-filtration 크로마토그라피를 이용한 천연분자량 분석결과, 67, 16.1 및 12.7 kDa의 3개 subunit가 3량체로 회합되어 존재하는 것으로 나타났으며 catalytic unit 당 2.2개의 니켈 원소를 함유하는 것으로 측정되었다. 정제된 효소의 비활성은 993.8 U/mg, <TEX>$K_m$</TEX>값은 8.5mM로 각각 산출되었다. Staphylococcus epidermidis is a coagulase-negative, gram-positive bacterium that normally inhabits the human skin. S. epidermidis is also known to be an opportunistic pathogen in infections of various indwelling medical devices. This report describes purification and characterization of the urease of S. epidermidis urease, which may act as a virulence factor. The urease from S. epidermidis was purified 1,127 fold by using DEAE-Sepharose, Phenyl-Sepharose, Mono-Q and Superdex HR200 column chromatography. The specific activity of the purified enzyme was 993.8 U/mg. Michaelis constant(<TEX>$K_m$</TEX>) of the enzyme was estimated to be 8.5 mM urea by using Lineweaver-Burke double reciprocal plot. The native molecular weight of the urease was shown to be 255 kD by using Superose 6HR gel filtration chromatography and the purified enzyme contained 2.2 nickel ions per catalytic unit. The overall stoichiometry of the enzyme subunits appears to be <TEX>$(\alpha\beta\gamma)_3$</TEX>, which is consistent with the enzymes from other bacteria sources.

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

Purification and Partial Characterization of Acetic Acid Esterase from Malted Finger Millet (Eleusinecoracana,Indaf-15)

Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme from cereals has not received much attention. In the present study, acetic acid esterase from 72 h ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatography, with a recovery of 0.36% and a fold purification of 34. The products liberated from alpha-NA and PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and 1H NMR. The pH and temperature optima of the enzyme were found to be 7.5 and 45 degrees C, respectively. The enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 degrees C. The activation energy of the enzymatic reaction was found to be 7.29 kJ mol-1. The apparent Km and Vmax of the purified acetic acid esterase for alpha-NA were 0.04 microM and 0.175 microM min-1 mL-1, respectively. The molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and metal ions such as Fe+3 and Cu+2 increased the activity while Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2, Zn+2, and Al+3 reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine was found to be a competitive inhibitor.

Purification and Characterization of a Cl−-Activated Aminopeptidase from Bovine Skeletal Muscle

To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-beta-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.

C-terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

Two Proteins, Mn2+, and Low Molecular Cofactor Are Required for C-Glucosyl-Cleavage of Mangiferin

C-Glucosides, in which sugars are attached to the aglycone by carbon-carbon bonds, are generally resistant to acid and enzyme hydrolysis. The C-glucosyl bond of mangiferin, a xanthone C-glucoside, was cleaved by anaerobic incubation with a human intestinal bacterium, Bacteroides sp. MANG, to give norathyriol. A cell-free extract obtained by sonication of B. sp. MANG demonstrated cleaving activity for mangiferin to norathyriol by adding NADH, diaphorase, and dithiothreitol. Both high molecular weight (>10 k) and low molecular weight (<10 k) fractions obtained from the cell-free extract were required for the activity. MnCl2 was necessary for the activity, but other metal ions were not. By purification of the high molecular weight fraction using DEAE-cellulose and Phenyl Sepharose column chromatography, two fractions, designated as proteins A and B, were separated and required for the activity. Neither protein A nor protein B alone showed any activity. This is the first report describing a C-glucosyl-cleaving enzyme from human intestinal bacterium that seems to involve a novel enzyme mechanism.

Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52: enhanced production and simple purification

An investigation was carried out on the enhancement of protease production and simple purification of an oxidant and SDS-stable alkaline protease produced by Bacillus clausii I-52 of industrial significance. The supplementation with 0.4% (w/v) NaCl and 0.05% (w/v) FeSO4.7H2O in a culture medium caused an increase in the protease production. The enzyme was purified to homogeneity with overall recovery of 79% and 10-fold purification from culture supernatant using Diaion HPA75, phenyl-Sepharose and DEAE-Sepharose column chromatographies. The protease was a halo-tolerant enzyme with apparent molecular mass of 28 kDa, and the Km and kcat values for N-Succinyl-Ala-Ala-Pro-Phe-pNA at 45 degrees C and pH 11.0 were determined to be 83.9 micromol l(-1) and 238.6 s(-1) respectively. Bacillus clausii I-52 was identified as a halo-tolerant bacterium, and the extracellular alkaline protease produced by B. clausii I-52 also showed extreme halo-tolerance. The enzyme stability towards SDS and H2O2 could be increased by adding NaCl or propylene glycol to the enzyme solution. The alkaline protease secreted by B. clausii I-52 is significant from an industrial perspective because of its stability against surfactants and oxidants as well as its tolerance towards high salinity. These enzymatic properties suggest its suitable application for industrial purposes.

Application of a thermostable glutamate racemase from Bacillus sp. SK-1 for the production of d-phenylalanine in a multi-enzyme system

A gene encoding glutamate racemase (GluRA) was found in a thermophilic Bacillus strain named SK-1. The gene was cloned and expressed in Escherichia coli WM335, a d-glutamate auxotroph. It consists of 792 bp with a start codon, TTG. The amino acid sequence deduced from the gene indicates that the GluRA has two cysteines and their surrounding regions are well conserved. The GluRA produced in the recombinant E. coli was purified to homogeneity by heat-treatment and Resource Q and Phenyl sepharose column chromatographies. The enzyme, which was determined to be a monomeric protein with a molecular weight of 29,000, did not require a cofactor such as pyridoxal 5′-phosphate, nicotinamide, or flavin for its activity. The enzyme was stable after incubation at 55 °C and retained 60% of its original activity after incubation at 60 °C. It was found to be stable in the region of pH 6.0–11.5. The thermostable GluRA was used as a catalyst in a multi-enzyme system composed of four enzyme reactions for the production of d-phenylalanine. By running the multi-enzyme system for 35 h, 58 g l −1 of d-phenylalanine was produced with 100% of optical purity from equimolar amount of phenylpyruvate.

Purification and properties of extracellular phytase from Bacillus sp. KHU-10.

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40 degrees C without 10 mM CaCl2 and pH 6.0-9.5 and 60 degrees C with 10 mM CaCl2. About 50% of its original activity remained after incubation at 80 degrees C or 10 min in the presence of 10 mM CaCl2. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The Km value for sodium phytate was 50 microM. Its activity was inhibited by EDTA and metal ions such as Ba2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, and Mn2+ ions.

7] Purification and properties of Rab3 GDP/GTP exchange protein

This chapter describes the assays for Rab3 guanine nucleotide exchange protein (GEP) activity, the procedures for the purification of native Rab3 GEP from rat brain, the procedures for the purification of recombinant Rab3 GEP from COS7 cells, and the properties of Rab3 GEP. The Rab3 GEP activity to stimulate the GDP/GTP exchange reaction of Rab3A is assayed by measuring either the dissociation of [ 3 H]GDP from or binding of [ 35 S]GTPTS to lipid-modified Rab3A. The steps used in the purification of Rab3 GEP from rat brain are: (1) preparation of the synaptic soluble fraction from rat brain, (2) Q-Sepharose FF column chromatography, (3) phenyl-Sepharose column chromatography, (4) hydroxyapatite column chromatography, (5) Mono Q HR10/10 column chromatography, (6) HiLoad 16/60 Superdex 200 column chromatography, and (7) HPLC hydroxyapatite column chromatography. All the purification procedures are carried out at 0–4°C.

Effect of ionic concentration on the higher-order structure of prophenol oxidase in Drosophila melanogaster.

Phenol oxidase in Drosophila melanogaster occurs as precursors designated prophenol oxidases A1 and A3. Crossing experiments between isozyme variants proved that prophenol oxidase in this species is a homodimer. Prophenol oxidases were partially purified using ammonium sulfate fractionation, phenyl Sepharose, and DEAE-cellulose column chromatography. The preparations were mixed, then dialyzed against buffer containing varying salt concentrations. The resulting prophenol oxidase was analyzed by gel electrophoresis. At 20 mM KCl or NaCl, two bands of phenol oxidase were observed, corresponding to the parental ones as monomer, whereas at 200 mM KCl or NaCl, three bands appeared in the gel, one being a dimer. The monomer-dimer reversibility of the Drosophila prophenol oxidase depends on the salt concentrations. The phenol oxidase activity remained unaffected within the KCl concentrations tested. Considering the ionic concentration of Drosophila hemolymph, these results indicate that prophenol oxidase exists as a dimer in vivo, and the higher-order structure of prophenol oxidase can be altered reversibly by ionic concentrations in vitro.