Effect of the modification of carboxylic groups in protohemin IX on the properties of endoperoxide prostaglandin synthetase

The effect of protohemin IX and its modified analogs (monomethyl ester, dimethyl ester, as well as monoamides with Val-Phe-OCH3 or Leu-His-OCH3) has been examined on the activity of prostaglandin endoperoxide synthetase from sheep vesicular glands (PGH-synthetase, EC 1.14.99.1, isolated as apoenzyme). For holoenzymes having the above compounds as prosthetic groups, the dissociation constants, relative activities and the apparent inactivation constants in the course of the reaction have been determined. The effect of Tween 20 on the indicated parameters for holoenzymes with protohemin IX and its mono- and dimethyl esters has been studied. Modification of one of the two carboxylic groups of protohemin IX markedly increases the dissociation constant for the respective holoenzyme and virtually does not affect catalytic activity. Modification of both carboxylic groups of protohemin IX hinder the binding with the apoenzyme and strongly reduces the catalytic activity of the holoenzyme.     

Correlation between hemin content and the chemiluminescent luminol reaction with bacteria.

Protohemin and covalently bound hemin were determined in eight aerobic bacterial strains. A good correlation between protohemin content and luminol reactivity was found. The ratio of luminol reaction to protohemin for the eight investigated strains was essentially identical to that of pure protohemin, 0.7 X 10(16) mV/mol. Covalently bound hemin contributed to the chemiluminescence to a minor extent only (0.7 X 10(14) mV/mol, in accordance with earlier observations of the lower reactivity of cytochrome c and related compounds. A difference in reaction kinetics of the luminol reaction with covalently bound hemin (slower reaction than protohemin) and protohemin was observed in vivo as well as in vitro. The phenomenon could be used to differentiate between strains with different hemin composition.     

Correlation between hemin content and the chemiluminescent luminol reaction with bacteria.

Protohemin and covalently bound hemin were determined in eight aerobic bacterial strains. A good correlation between protohemin content and luminol reactivity was found. The ratio of luminol reaction to protohemin for the eight investigated strains was essentially identical to that of pure protohemin, 0.7 X 10(16) mV/mol. Covalently bound hemin contributed to the chemiluminescence to a minor extent only (0.7 X 10(14) mV/mol, in accordance with earlier observations of the lower reactivity of cytochrome c and related compounds. A difference in reaction kinetics of the luminol reaction with covalently bound hemin (slower reaction than protohemin) and protohemin was observed in vivo as well as in vitro. The phenomenon could be used to differentiate between strains with different hemin composition.     

Thermal denaturation and regeneration of japanese-radish peroxidase.

Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin.     

The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.     

Synthesis of arginine-containing peptides and their conjugates with protohemin IX and tetraphenylporphyrin

Arg-containing peptides and their conjugates with protohemin IX were synthesized by the solid phase method using Merrifield resin. The conjugates of Arg-containing peptides with tetraphenylporphyrin were obtained by using phosphorus trichloride as an activating agent.     

ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein.

Electron nuclear double resonance (ENDOR) signals have been obtained from iron-linked nitrogens in frozen solutions of cytochrome c, metmyoglobin cyanide, and a low spin protohemin mercaptide complex. Hyperfine couplings from heme protons have also been obtained from metmyoglobin cyanide and from a low spin protohemin cyanide complex. Several of these proton resonances are assigned to specific heme protons.     

Esterification of the propionate groups promotes alpha/beta hemoglobin chain homogeneity of CN-hemin binding

This study examines the post-translational role of peripheral propionate groups in the incorporation of the Fe-protoporphryin IX heme into nascent alpha- and beta-globin chains. Human apohemoglobin (a heme-free alpha/beta dimer) in 0.05 M potassium phosphate buffer, pH 7, at 20 degrees C was titrated with either CN-protohemin (native heme with two peripheral propionate groups), or CN-dimethylester hemin (a modified heme with two methyl ester groups in place of the propionate groups). Soret spectrophotometric CN-hemin titrations confirmed that a spectral shift resulted upon binding of protohemin, but no spectral shift occurred upon binding the dimethylester derivative. Recent studies have correlated a Soret spectral shift with the preferential heme binding to the alpha subunit of apohemoglobin. The absence of a Soret wavelength shift (in conjunction with molecular modeling) presented here suggested that the modification of heme propionate groups prevented the formation of an alpha-heme/beta-globin intermediate, a requisite step in the normal assembly of functional hemoglobin.     

Purification, crystallization, and characterization of peroxidase from Coprinus cinereus

Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S.F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41,600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.     

1H NMR Study of the influence of hemin vinyl-->methyl substitution on the interaction between the C-terminus and substrate and the "aging" of the heme oxygenase from Neisseria meningitidis: induction of active site structural heterogeneity by a two-fold symmetric hemin

Solution 1H NMR has been used to characterize the active site molecular and electronic structure of the cyanide-inhibited 2,4-dimethyldeuterohemin complex of the heme oxygenase from Neisseria meningitidis (NmHO) with respect to the mode of interaction of the C-terminus with the substrate and the spontaneous "aging" of NmHO that results in the cleavage of the C-terminal Arg208-His209 dipeptide. The structure of the portion involving residues Ala12-Phe192 is found to be essentially identical to that of the protohemin complex in either solution or crystal. However, His207 from the C-terminus is found to interact strongly with the substrate 1CH3, as opposed to the 8CH3 in the protohemin complex. The different mode of interaction of His207 with the alternate substrates is attributed to the 2-vinyl group of protohemin sterically interfering with the optimal orientation of the proximal helix Asp27 carboxylate that serves as acceptor to the strong H-bond by the peptide of His207. The 2,4-dimethyldeuterohemin HO complex "ages" in manner similary to that of protohemin, (Liu, Y., Ma, L.-H., Satterlee, J.D., Zhang, X., Yoshida, T., and La Mar, G. N., (2006) Biochemistry 45, 3875-3886) with mass spectrometry and N-terminal sequencing indicating that the Arg208-His209 dipeptide is cleaved. The 2,4-dimethyldeuterohemin complex of WT HO populates an equilibrium isomer stabilized in low phosphate concentration for which the axial His imidazole ring is rotated by approximately 20 degrees from that in the WT. The His ring reorientation is attributed to Asp24 serving as the H-bond acceptor to the His207 peptide NH, rather than to the His23 ring NdeltaH as in the crystals. The functional implications of the altered C-terminal interaction with substrate modification are discussed.     

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis. A 1H NMR study

Heme oxygenase (HO), from the pathogenic bacterium N. meningitidis(NmHO), which secures host iron, shares many properties with mammalian HOs but also exhibits some key differences. The crystal structure appears more compact, and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D (1)H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross-peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin, and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~10(2) increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed.     

Characterization of the spontaneous "aging" of the heme oxygenase from the pathological bacterium Neisseria meningitidis via cleavage of the C-terminus in contact with the substrate. Implications for functional studies and the crystal structure

Solution 1H NMR spectroscopy and mass spectrometry are utilized to characterize the irreversible "aging" of native heme oxygenase from N. meningitidis, NmHO. 2D NMR characterization of the cyanide-inhibited substrate complex shows that the C-terminal interaction between Arg208His209 and the exposed pyrrole of the protohemin substrate in the "native" NmHO complex is lost in the "aging". Mass spectrometry and N-terminal sequencing of wild type and "aged" NmHO reveal that the "aging" process involves cleavage of the Arg208His209 dipeptide. The construction of the double deletion mutant without Arg208His209 and its NMR comparison as both the resting state substrate complex and its cyanide-inhibited complex with the "aged" NmHO reveal that cleavage of the C-terminal dipeptide is the only modification during the aging. Comparison of cyanide ligand binding constants reveal a factor approximately 1.7 greater CN- affinity in the native than "aged" NmHO. The rate of protohemin degradation and its stereoselectivity are unaffected by the C-terminal truncation. However, the free alpha-biliverdin yield in the presence of desferrioxamine is significantly increased in the "aged" NmHO and its deletion mutant relative to WT, arguing for a role of the NmHO C-terminus in modulating product release. The facile cleavage of Arg208His209 in the resting state complex, with a half-life of approximately 24 h at 25 degrees C, suggests that previous characterization of NmHO may have been carried out on a mixture of native and "aged" NmHO, and may account for the "lost" C-terminal residues in the crystal structures.     

Purification and characterization of a cationic peroxidase Cs in Raphanus sativus

A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase Cs was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase Cs was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase Cs with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase Cs has Km values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase Cs showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination.     

Spin label studies on conformational changes of aphohemoglobin due to heme binding.

Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.     

Effect of the structure of the prosthetic group on the catalytic properties of prostaglandin H synthetase

The effect of vinyl groups of protohemin IX on its cofactor properties with respect to prostaglandin H synthetase has been studied. It was shown that substitution of ethyl groups or a hydrogen for vinyl groups affects neither binding of the prosthetic group to the apoenzyme nor catalytic properties of holo-prostaglandin H synthetase. Replacement of vinyl groups with bulkier substituents (hydroxyethyl or acetyl groups) decreases holoenzyme stability and catalytic activity. By comparison of the cofactor properties of protoporphyrin and hematoporphyrin macrocycles with different central ions (Fe3+, Mn2+, 2H+ in the case of protoporphyrin, and Fe3+, Mg2+, Cd2+ and Cu2+ in the case of hematoporphyrin), the presence of Fe3+ ions was shown to be mandatory for prostaglandin H synthetase activity. It was demonstrated that the cofactor structure modifications do not affect the holo-prostaglandin H synthetase inactivation rate constant in a reaction.     

Diffuse reflectance infrared Fourier transform spectroscopic characterization of a silica-immobilized N-hydroxysuccinimide active ester crosslinking agent and its precursors

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.     

Reversible oxygenation of protoheme-imidazole complex in aqueous solution (1,2)

Solutions of protohemin in aqueous buffer containing imidazole were reduced and exposed to carbon monoxide forming the carbon monoxide-imidazole complex similar to that in carboxyhemoglobin. This complex is stable for long periods in the presence of low pressures of oxygen and thus the standard flash photolysis methods can be used to determine rates of combination of the heme-imidazole complex with oxygen. Combination rates for both carbon monoxide and oxygen are faster than any on rates for hemoglobin and oxygen dissociation rates are also faster. But the equilibrium constant for binding of this isolated site is larger than that for hemoglobin.     

Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.     

A method for the estimation of thiol esters.

1. A method is described for the estimation of thiol ester groups. The thiol ester is converted into the corresponding thiol by reaction with ammonia; the thiol is then titrated amperometrically with mercuric chloride. 2. The method may be used in the presence of SH and S.S groups. The SH groups are titrated at pH3 in the presence of excess of chloride; under these conditions thiol esters do not react with mercuric chloride. Thiol ester plus thiol is then estimated by titration after reaction with ammonia. Finally, titration after reaction with ammonia and sulphite gives the thiol ester plus thiol plus disulphide. 3. The procedure has been applied to glyceraldehyde phosphate dehydrogenase. The enzyme was found to contain 15-16 SH groups/mol. and no S.S groups. After reaction with acetyl phosphate 1.8-3.5 thiol ester groups were detected, the number depending on the conditions of acetylation. In the absence of bound NAD, the number of thiol ester groups formed was 1.8/mol., although a value of 2.9 labile acetyl groups/mol. was given by the method of Lipmann & Tuttle (1945). The presence of thiol ester groups in the S-(d-3-phosphoglyceryl)-enzyme was also demonstrated.     

左旋多巴甲酯在PLGA微球的稳定性研究

目的：考察左旋多巴甲酯在PLGA微球中的稳定性并探讨其稳定方法。方法：利用HPLC的方法考察了左旋多巴甲酯在不同pH值和光照的环境中和微球里的稳定性。结果：左旋多巴甲酯在pH3中稳定,在微球中也可以稳定一周。结论：包封左旋多巴甲酯在PLGA微球中,是一种有效地保护了左旋多巴甲酯在微球中的活性,可以实现长效缓释,是一种可行的方案。