Oxidative cleavage of DNA by a dipyridoquinoxaline copper(II) complex in the presence of ascorbic acid

Complex [Cu(dpq)(2)(H(2)O)](ClO(4))(2).H(2)O (1), where dpq is dipyrido-[3,2-D:2',3'-f]-quinoxaline, has been prepared by reacting copper(II) perchlorate hexahydrate with dpq in methanol and structurally characterized. The complex crystallizes in the triclinic space group P-1 with the unit cell parameters a=8.646(2) A, b=12.290(5) A, c=14.283(4) A, alpha=94.01(2) degrees, beta=91.69(2) degrees,gamma=101.60 (3) degrees, V=1481.7(8) A(3) and Z=2. The structure, refined to R=0.0505 and R(w)=0.1441 for 5212 reflections with I>2sigma (I) using 440 parameters, shows the presence of a CuN(4)O chromophore in an axially compressed distorted trigonal-bipyramidal structure. The Cu-N distances lie in the range 1.969(3)-2.103(3) A. The Cu-OH(2) distance is 2.145(3) A. The complex is one-electron paramagnetic and exhibits a visible spectral d-d band at 718 nm in MeCN. It shows a quasi-reversible cyclic voltammetric response at 0.091 V (DeltaE(p)=229 mV) at 50 mV s(-1) in MeCN-0.1 M TBAP for the Cu(II)/Cu(I) couple. In 50 mM Tris-HCl/0.1 M KCl buffer-DMF mixture (1:4 v/v, pH 7.2), the couple appears at 0.089 V versus SCE. The complex undergoes facile reduction with sodium ascorbate in an aqueous DMF mixture (4:1 v/v) to form an unstable brown Cu(I) species (lambda(max)=440 nm, epsilon=7480 M(-1) cm(-1)) which converts to 1 on exposure to air giving a turnover frequency of ca. 400. Binding studies revealed that 1 is an efficient binder to calf thymus DNA. Complex 1 on reaction with supercoiled (SC) DNA in presence of ascorbic acid in a 50 mM Tris-HCl/50 mM NaCl buffer (pH 7.2) shows nuclease activity which is 4.5 times greater than that of the phen analogue.     

Effect of N-bromosuccinimide-modification of tyrosine side chains of cardiotoxin II of the Indian cobra on biological activity

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4.0), 0.01 N H₂SO₄ (pH 2.0) and Tris-HCl buffer (pH 8.5) on oxidation of tyrosine residues was exa mined. In comparison with 0.01 N H₂S O₄, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodo-succinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3.75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.     

Mixed ligand copper(II) complexes of phenanthroline/bipyridyl and curcumin diketimines as DNA intercalators and their electrochemical behavior under Nafion and clay modified electrodes

The mode of binding of copper(II) mixed ligand complexes of phen/bpy and Knoevenagel condensate of curcumin (4-salicylidene-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) and 4-X-anilines with herring sperm DNA has been investigated using spectral and electrochemical techniques in Tris-HCl buffer pH 7.1. On titration with DNA, usual hypochromism and unusual (large) red shift (30-35 nm) for some of these complexes were observed in their absorption spectra of intense intraligand (IL) pi-pi* transition around 420 nm. Variations in the absorbance due to their interaction with DNA on time scale were also investigated, under fixed concentrations of complex and DNA. On interaction with DNA, the quasi-reversible CuII/I redox couple slightly improves its reversibility with considerable decrease in current intensity. The intercalation of these copper complexes into the DNA base pairs was also investigated by gel retardation assay method. All the experimental results indicate that the phen mixed copper(II) complexes intercalate more effectively into the DNA base pairs than their bpy counterparts. Significant differences in the redox behavior of these copper(II) complexes under electrochemically modified GC electrodes with Nafion and K10 Montmorillonite clay have also been investigated and discussed.     

Oxidative DNA damage induced by HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer in the presence of Au(III)

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)](5+) and [Pd(II)(TMPyP)](4+), where [H(2)(TMPyP)](4+) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.     

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.     

Ultraviolet difference spectroscopy of bovine carbonic anhydrase substituted with various divalent metals

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5–9 are reported. For Zn(II) at all pH's and Co(II) at pH ? 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280–292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.     

Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.     

Spin-trapping study on the hydroxyl radical formed from a tea catechin-Cu(II) system

A spin-trapping method was applied to examine the formation of the hydroxyl (OH) radical from a tea catechin-Cu(II) system to elucidate a previous result that some tea catechin-Cu(II) systems induced DNA scission. Three tea catechins, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC), were used. The spin-trapping agent, 5,5'-dimethyl-pyrroline-1-oxide (DMPO), was dissolved in a pH 9 phosphate buffer solution, then a catechin and Cu(II) were added in that order, and the ESR spectral change was monitored for one hour. The order of adding the catechin and Cu(II) was then reversed, and the ESR spectral change was again monitored to examine the coordinating activity of each catechin toward the Cu(II) ion and the effect on OH radical generation. The intensity changes of the spin adducts, DMPO-OH, DMPO-CH3 and DMPO-H, were analyzed, the results suggesting that the OH radical generated in the system decomposed DMPO, resulting in the formation of DMPO-CH3 and DMPO-H. The results show that EGC formed a stable complex with Cu(II) and generated the OH radical. EGCg seemed to have this activity, but the OH radical that was generated was scavenged by the gallate group existing in the complex. EC did not show strong coordinating and OH-generating activities. These characteristics of the three catechins are consistent with the results shown for DNA scission.     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

The relation between carbon monoxide binding and the conformational change of hemoglobin.

The spectral difference between normal and rapidly reacting deoxyhemoglobin (Sawicki and Gibson (1976), J. Biol Chem. 251:1533-1542) is used to study the relationship between CO binding to hemoglobin and the conformational changes to the rapidly reacting form in a combined flow-laser flash experiment. In both pH 7 phosphate buffer and pH 7 bis(2-hydroxy-ethyl)imino-tris (hydroxymethyl)methane buffer (bis-Tris) with 500 muM 2,3-diphosphoglycerate (DPG), the conformational change lags far behind CO binding; rapidly reacting hemoglobin is not observed until more than 10% of the hemoglobin is liganded. In pH 9 borate buffer the formation of rapidly reacting hemoglobin leads CO binding by a significant amount. A simple two-state allosteric model (Monod et. al. (1965), J. Mol. Biol. 12:88-118) which assumed equivalence of the hemoglobin subunits in their reaction with CO was used to simulate the experimental results. In terms of the model, the conformational change lead observed at pH 9 suggests that significant conformational change has occurred after binding of only one CO molecule per tetramer. In the presence of phosphates good agreement between experimental results and simulations is obtained using parameter values suggested by previous experimental studies. The simulations suggest that the conformational change occurs after binding of three CO molecules.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.     

Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II.

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.     

Paradoxical role of lipid metabolism in heart function and dysfunction

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effecive than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(1)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.     

Association of tubulinyl-tyrosine carboxypeptidase with microtubules

Evidence is presented that the topoisomerase inhibitors novobiocin and coumermycin inhibit the production of double-strand breaks in mouse mastocytoma cell nuclear DNA by the anticancer drug 4'[(9-acridinyl)amino]-methanesulphon-m-anisidide (mAMSA). Novobiocin did not inhibit resealing of DNA breaks induced by mAMSA. It is suggested that mAMSA intercalation into DNA induces the action of a type II topoisomerase. mAMSA and oAMSA were equally effective in breaking the DNA in isolated nuclei.     

Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide

Oxidative DNA damage has been implicated in diverse biological processes including mutagenesis, carcinogenesis, aging, radiation effects, and chemotherapy. We examined the in vitro effect of low concentrations of Cu(II) or H2O2 alone and in combination on supercoiled plasmid DNA. As much as 10(-2) M Cu(II) or 10(-2) M H2O2 alone did not break the DNA. However, a mixture of 10(-6) M Cu(II) plus 10(-5) M H2O2 produced strand breaks and inactivated transforming ability. Strand breakage was proportional to incubation time, temperature, and Cu(II) and H2O2 concentrations. Abasic sites were not detected. Strand breakage was inhibited by metal chelators, catalase, and by high levels of free radical scavengers implying that Cu(II), Cu(I), H2O2, and .OH were involved in the reaction. The extent of DNA strand breakage was not affected by superoxide dismutase indicating that superoxide was not a major contributor to the DNA damage. DNA sequence analysis demonstrated that hot piperidine-sensitive DNA lesions were produced preferentially at sites of 2 or more adjacent guanosine residues. This sequence specificity was observed with Cu(II) plus H2O2 but not with Cu(I) alone. Polyguanosine sequence specificity for DNA damage induction appears to be unique among simple chemical systems. This reaction may be important in mechanisms of oxidative damage in vivo.     

Oxidative cleavage of DNA by homo- and heteronuclear Cu(II)-Mn(II) complexes of an oxime-type ligand

Novel homodinuclear Cu(II) (K1), heterodinuclear Cu(II)-Mn(II) (K2) and homotrinuclear Cu(II) (K3) complexes with a novel oxime-type ligand have been prepared and their nucleolytic activities on pCYTEXP were established by neutral agarose gel electrophoresis. The analyses of the cleavage products obtained electrophoretically indicate that although the examined complexes induces very similar conformational changes on supercoiled DNA by converting supercoiled form to nicked form than linear form in a sequential manner as the complex concentration or reaction period is increased, K3 is less effective than the two others. The oxime complexes were nucleolytically active at physiological pH values but the activities of K1 or K2 were diminished by increasing the pH of the reaction mixture. In contrast, K3 makes dominantly single strand nicking by producing nicked circles on DNA at almost all the applied pH values. Metal complex induced DNA cleavage was also tested for inhibition by various radical scavengers as superoxide dismutase (SOD), azide, thiourea and potassium iodide. The antioxidants inhibited the nucleolytic acitivities of the oxime complexes but SOD afforded no protection indicating that the nucleolytic mechanism involves of copper and/or manganese complex-mediated reactive oxygen species such as hydroxyl radicals being responsible for the oxidative DNA cleavage.     

Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11

A copper [Cu(II)]-accumulating strain, Pseudomonas putida II-11, isolated from electroplating effluent removed a significantly high amount of Cu(II) from growth medium and buffer. A laboratory-scale fixed bed reactor with cells of P. putida II-11 immobilized in polyacrylamide gel was constructed. The adsorption of Cu(II) by the immobilized cells was pH-dependent. Maximum removal of Cu(II) by the immobilized cells was at pH 8.0. The presence of Cr(IV), Ni(II) and Zn(II) did not significantly inhibit Cu(II) uptake whereas the presence of Pb(II) reduced Cu(II) uptake by fivefold. The presence of borate, carbonate, chloride and sulphate did not significantly inhibit Cu(II) uptake. The Cu(II) removal capacity of the bioreactor with immobilized cells did not change significantly when operated at retention times greater than 3 min. More than 90% of Cu(II) adsorbed on immobilized cells could be recovered by eluting with 0.1 m HCl. The bioreactor could be used for at least five loading-elution cycles without loss of Cu(II) removal capacity. The feasibility of using this bioreactor to remove and recover Cu(II) from electroplating effluent is discussed. Correspondence to: P. K. Wong     

Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction

A novel and sensitive resonance scattering (RS) spectral immunoassay for the determination of microalbumin (Malb) was developed, based on the catalytic effect of immunonanogold (ING) probe on Fehling reagent-glucose reaction, and resonance scattering effect of Cu(2)O particles. Nanogold particles in size of 10nm were used to label goat anti-human microalbumin (GMalb) to obtain an ING probe (AuGMalb) for Malb. The probe produced unspecific aggregation in pH 5.0 citric acid-Na(2)HPO(4) buffer solutions. Upon addition of Malb, the dispersed ING complex formed. The ING complex in supernatant was obtained by centrifuging and was used as catalyst for the reaction between Fehling reagent and glucose to form the Cu(2)O particles to amplify the resonance scattering signal at 610 nm. With addition of Malb, the ING complex in the supernatant increased and the RS intensity at 610 nm (I(610 nm)) enhanced linearly. The enhanced intensity DeltaI(610 nm) was proportional to the Malb concentration in the range of 0.014-0.43 ng ml(-1), with a detection limit of 7.2 pg ml(-1). The proposed method was applied to detect Malb in human urine sample with satisfactory results.     

Reversible cyclization ofS-(2-oxo-2-carboxyethyl)-L-homocysteine to cystathionine ketimine

Summary S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N₂ atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer.Abbreviations CK cystathionine ketimine - OCEHC S-(2-oxo-2-carboxyethyl) homocysteine - HPLC high performance liquid chromatography     

Leghemoglobin. Low temperature optical spectra of acid and alkaline forms of leghemoglobin(IV). Configuration of the heme.

Leghemoglobin(IV), the derivative of leghemoglobin at the formal oxidation state IV, when cooled to liquid nitrogen temperature exhibits radically different spectra at acid and alkaline pH. The acid and alkaline forms are freely interconvertible. The optical spectrum of the acid form is closely similar to optical spectra of the red higher oxidation states of horseradish and cytochrome c peroxidases, showing that the configuration of the heme iron is the same throughout this family of compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectrum of the alkaline form of leghemoglobin(IV) recalls that of alkaline low spin ferric leghemoglobin. Near infrared spectra of leghemoglobin(IV), myoglobin(IV), and the higher oxidation states of the peroxidases are featureless to 1300 nm, suggesting a common structural feature. The acid form of leghemoglobin(IV), seen in fluid buffer as a transient species at pH 5 or less, is conveniently generated by cooling a solution of the more stable alkaline form in borate buffer to liquid nitrogen temperature. At this temperature borate buffers become acid.