Effect of indole-3-acetic acid on the kinetics of horseradish peroxidase catalyzed scopoletin oxidation

The kinetics of horseradish peroxidase catalyzed of scopoletin oxidation were observed to be sigmoidal. The apparent KmS for scopoletin is 0.9 mM. Inhibition of scopoletin oxidation by indole-3-acetic acid (IAA) appears to be non competitive. Non competitive inhibition by IAA suggests a more significant role of the peroxidase protein matrix in regulating the activity of the heme moiety.     

Esterase coupled with the H2O2/horseradish peroxidase system triggers chemiluminescence from 2-methyl-1-propenylbenzoate: a potential analytical tool for esterase analysis

Suppression of the fibrinolytic activity plays an important role in the prevention of hemorrhage during pregnancy and labor. A hypofibrinolytic and hypercoagulable state may be established in the placenta during pregnancy. However, little infraction is present in the normal placenta. This evidence shows that placenta maintains the fibrinolytic activity in spite of hypercoagulable state. As there is a high amount of APC in the placenta, APC is thought to be involved in fibrinolysis of placenta. Thus, we studied the role of APC on fibrinolysis in placenta. (1) uPA activity of cell membrane reappears after incubation with uPA/PAI-1 complex and a large amount of APC by flow cytometry, (2) APC was made PAI-1/APC complex after incubation of uPA/PAI-1 complex with APC. Our results suggest that APC is the important substance for fibrinolysis in the placenta by decreasing of PAI activity.     

Luminol chemiluminescence in rat macrophages and granulocytes: the role of NO, O2-/H2O2, and HOCl

Luminol chemiluminescence was increased up to five-fold by L-arginine and markedly inhibited by NG-nitro-L-arginine (L-NNA) in phorbol ester (PMA) or opsonized zymosan-activated rat Kupffer cells, and in PMA-activated rat peritoneal and alveolar macrophages. While in Kupffer cells these effects did occur without pretreatment with lipopolysaccharides (LPS), LPS pretreatment was a requirement in peritoneal and alveolar macrophages. Azide (0.05 mM) had no effect on luminol chemiluminescence in the macrophages. The changes in luminol chemiluminescence were accompanied by parallel changes in nitric oxide (NO) formation. Macrophage superoxide anion radical (O2-) production was not significantly changed by addition of L-arginine and L-NNA nor by pretreatment with LPS. No hypochlorous acid (HOCl) formation was detectable in the macrophages. In contrast, in rat granulocytes activated by a variety of stimuli including PMA, zymosan, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and the calcium ionophore A23187 with or without pretreatment with LPS, L-arginine and L-NNA had no effect on luminol chemiluminescence. Luminol chemiluminescence, however, was largely inhibited by 0.05 mM azide. The activated granulocytes released significant amounts of HOCl but did not generate NO. These results demonstrate that NO may largely contribute to luminol chemiluminescence in rat macrophages, in which HOCl formation does not occur. On the other hand, HOCl is the reactive oxygen species responsible for luminol chemiluminescence in rat granulocytes, where NO is formed only in minor quantities, if at all.     

Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I

A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Morever it has been possible to relate the reduction rate constants of monosubstituted substrates by a linear free-energy relationship (Hammett equation). The correlation of log (rate constants) with sigma values (Hammett equation) and the absence of correlation with sigma+ values (Okamoto-Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained for p-aminophenol indicates that a value of 2.3 X 10(8) M-1 S-1 probably approaches the diffusion-controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.     

trans-2-phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic 'donors' such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.     

Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.     

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.     

Potential cancerostatic benfluron is metabolized by peroxidase: in vitro biotransformation of benfluron by horseradish peroxidase

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of cytokines. The principal function of MCP-1 is thought to be the stimulation of monocyte recruitment. Monocyte products are potential regulators of bone cell activity. Growth factors produced by monocytes may stimulate bone formation, while cytokines such as IL-1 and IL-6 can induce bone resorption. To determine whether MCP-1 enhances recruitment of monocytes during bone healing, studies were carried out in which MCP-1 was applied to osseous sites in vivo. Changes in monocyte number were determined by immunohistochemistry using the antibody ED-1 specific for peripheral monocytic cells. The effect of MCP-1 on osteoblast number was determined by counting the number of alkaline phosphatase positive cells in close proximity to bone. For comparison, osteoblast number was also determined following stimulation with platelet-derived growth factor (PDGF)-BB plus IGF-1 in vivo. Results indicate that MCP-1 stimulated a large increase in monocyte recruitment compared to vehicle alone. An increase in monocytes induced by MCP-1 was associated with an increase in the number of osteoblasts lining the bone surface, although not to the same magnitude as a positive control, PDGF-BB, and IGF-1. These results indicate that MCP-1 induces the recruitment of monocytes to bone and suggest that the recruitment is associated with an increase in osteoblast number. This is likely to occur via indirect mechanisms, because MCP-1 did not directly enhance DNA synthesis in osteoblastic cells in vitro. Thus, activated mononuclear phagocytes may play an important role in osseous wound healing by stimulating proliferation of osteoblastic cells, presumably through the elaboration of growth factors.     

Lipoxygenase/H2O2-catalyzed oxidation of dihdroxyindoles: synthesis of melanin pigments and study of their antioxidant properties

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H2O2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.     

LaCl3·7H2O晶体的热分解

采用 TG- DTG方法 ,得到 L a Cl3 · n H2 O晶体在空气中热分解过程及相应分解反应的活化能 ,即在84～ 112℃时 ,L a Cl3 · 7H2 O失去 4个结晶水 ,分解反应的活化能为 133.2 k J/ mol,在 12 3.2～ 137.9℃时 ,L a Cl3 ·3H2 O失去 2个结晶水 ,分解反应的活化能为 15 3.9k J/ mol,在 16 2 .5～ 177.4℃时 ,L a Cl3 · H2 O失去 1个结晶水 ,分解反应的活化能为 45 4.6 k J/ m ol,在 397.7～ 46 4.5℃时 ,L a Cl3 气相水解为 L a OCl,活化能为 6 6 k J/ m ol。在温度低于90 0℃ (最高测试温度 )时 ,L a OCl能稳定存在。     

钴催化过氧化氢氧化二溴茜素紫的反应动力学及其应用

基于在pH11.92的Brittom-Robinson缓冲溶液中,钴?催化过氧化氢氧化二溴茜素紫的反应,建立了测定痕量钴的催化光度新方法.研究了反应条件,确立了动力学方程,测定了动力学参数,探讨了反应机理.方法的检出限是1.2×10-11g/mL,测定范围是0.00～1.0μg/mL,方法用于井水、蜂蜜、维生素B12中痕量钴的测定,结果满意.     

Intracellular staining of Purkinje cells and their axons with horseradish peroxidase

Neuroendocrine assessments were made in a 16-year-old girl with pseudocyesis of 38 weeks duration. Basal levels of pituitary LH and PRL, but not of FSH, were markedly elevated and the pulsatile pattern of LH and PRL appears to be exaggerated compared with those found in normal cycling women. Growth hormone levels were normal, with an appropriate increase after arginine infusion and l-dopa. The increased luteotropic action resulting from increased plasma LH and the lactogenic action of increased plasma PRL were associated with the presence of luteal function and galactorrhea. The dominant role of psychic mechanism(s) in causing hypersecretions of LH and PRL is suggested by the almost immediate fall in the serum concentrations and the rapid resolution of abdominal distention after the diagnosis was revealed to the patient.     

过氧化氢氧化茜素绿动力学光度法测定痕量钴

基于在三乙醇胺活化剂存在下,钴 对过氧化氢氧化茜素绿反应的催化作用,提出一种高灵敏度测定痕量钴 的新方法。该法线性范围为8～56μg/L,检出限为0.34μg/L。测定出反应表观活化能Ea=41.86kJ/mol。该法用于茶叶及人发样品中钴的测定,获得满意结果。