Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.     

管碟法测定生物效价影响因素的探讨及改进

《中国兽药典》（2005年版）收载的兽用抗生素药品效价的微生物检定包括两种方法,即管碟法和浊度法。目前多数使用者仍以管碟法为主。管碟法作为抗生素效价测定的经典方法,具有它的优点,但此方法操作步骤复杂,试验影响因素多,需要从每个环节严格控制,才能得到与真值更为接近的结果。笔者结合日常工作实践,对管碟法测定抗生素生物效价过程中的多种影响因素进行探讨及改进,使测定条件得到了较好的控制,有利于提高效价测定结果的精密度和准确度。     

Variation in ptaquiloside content in bracken (Pteridium esculentum (Forst. f) Cockayne) in New Zealand

AIM: To examine stands of bracken fern (Pteridium esculentum) from throughout New Zealand for the presence and concentration of ptaquiloside (Pta), and to compare the presence and/or concentrations of Pta in areas where bovine enzootic haematuria (BEH) and/or acute haemorrhagic syndrome (AHS) has been known to occur with those where BEH/AHS has not been recorded.

METHODS: Stands of bracken fern were sampled from 275 sites throughout New Zealand. Sixty-two stands were from a regional survey predominantly from the Waikato and Coromandel regions, 27 were from a farm in the King Country where BEH/AHS had been investigated previously, and 186 were from a national survey of the North and South Islands. Sampling sites were from a mixture of grazed paddocks, roadsides, and forest and bush areas. Samples comprised whole young fronds, the tops of unfurling young fronds, or, for the Regional Survey, mature green fronds from the previous season. Pta was extracted from the samples, and measured using high-performance liquid chromatography. Information on the occurrence of BEH/AHS at specific locations was obtained from published information and records from animal health laboratories in New Zealand.

RESULTS: The 275 samples contained widely varying concentrations of Pta. In the Farm Survey, concentrations ranged from 280–13,300 (mean 3,800) µg/g (on a dry-weight basis) in the 63% of samples that contained Pta. A high proportion of samples from the Regional and National Surveys covering large areas of the country contained no detectable levels of Pta. The majority (61%) of samples from these two surveys which contained Pta were from areas where BEH/AHS was reported to occur. Combining data from all surveys, in areas with reported BEH/AHS, 42% of samples collected contained Pta, compared with 6% where BEH/AHS was not known to occur.

CONCLUSIONS: Concentrations of Pta in bracken in New Zealand vary greatly, and in a high proportion of stands Pta is not found. A higher incidence of Pta, and some very high concentrations, are found in areas where BEH/AHS was known to occur.     

Heme-mediated production of free radicals via preformed lipid hydroperoxide fragmentation

Electron spin resonance (ESR) spectroscopy and the spin-trapping technique were used to investigate the capacity of several hemoglobin (Hb) forms of rainbow trout (oxyHb and metHb), free hemin (oxidized form of heme group), and hemin complexed with bovine serum albumin (BSA) to promote formation of free radicals via fragmentation of preformed lipid hydroperoxides. Cumene hydroperoxide (CumOOH) was used as a model for lipid hydroperoxide, and free radicals were monitored by stabilizing with the spin traps alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Two different types of free radicals, hydroxyl and carbon-centered radicals, were identified as a result of the interaction of the heme-containing systems and CumOOH. Carbon-centered radicals were found to be mainly heme-mediated because the addition of the iron-chelating agent EDTA did not affect the formation of POBN/carbon-centered adducts. Hemin alone was the best promoter for the production of POBN/carbon-centered radicals in the presence of low hydroperoxide concentrations (below equimolar condition over heme group), whereas hemin/BSA and oxyHb were more active in generating radicals at high hydroperoxide concentrations or after successive interactions with hydroperoxides. This finding can be explained by the coexistence of two different facts: (i) the interaction between hemin and lipid hydroperoxides seems to be more efficient in the case of free hemin compared to heme-protein complexes and (ii) a faster degradation of hemin is produced without the presence of a protein fraction, globin or albumin. The comparison of oxyHb and metHb also suggested that both Hb redox states have similar capacities to generate oxidative stress via cleavage of preformed lipid hydroperoxides.     

The Interdependence of Deformational and Thermal Processes in Mountain Belts

Crustal temperatures within collisional orogens are anomalously high compared with temperatures at comparable depths in stable continents, which is evidence of thermal processes that are fundamental to orogenesis. These temperatures can be explained by the redistribution of crust enriched in heat-producing elements through the accretion of crust from the down-going plate to the upper plate and surface erosion. With the use of geologically reasonable rates, the model results predict high temperatures (over 600°C) and inverted upper-plate geotherms (about 100°C over 20 kilometers) at shallow depths (20 to 40 kilometers) by 25 to 35 million years after collision. This study emphasizes the interdependence of deformational, surficial, and thermal processes.     

Interactions between iron, phenolic compounds, emulsifiers, and pH in omega-3-enriched oil-in-water emulsions

The behavior of antioxidants in emulsions is influenced by several factors such as pH and emulsifier type. This study aimed to evaluate the interaction between selected food emulsifiers, phenolic compounds, iron, and pH and their effect on the oxidative stability of n-3 polyunsaturated lipids in a 10% oil-in-water emulsion. The emulsifiers tested were Tween 80 and Citrem, and the phenolic compounds were naringenin, rutin, caffeic acid, and coumaric acid. Lipid oxidation was evaluated at all levels, that is, formation of radicals (ESR), hydroperoxides (PV), and secondary volatile oxidation products. When iron was present, the pH was crucial for the formation of lipid oxidation products. At pH 3 some phenolic compounds, especially caffeic acid, reduced Fe(3+) to Fe(2+), and Fe(2+) increased lipid oxidation at this pH compared to pH 6. Among the evaluated phenols, caffeic acid had the most significant effects, as caffeic acid was found to be prooxidative irrespective of pH, emulsifier type, and presence of iron, although the degrees of lipid oxidation were different at the different experimental conditions. The other evaluated phenols were prooxidative at pH 3 in Citrem-stabilized emulsions and had no significant effect at pH 6 in Citrem- or Tween-stabilized emulsions on the basis of the formation of volatiles. The results indicated that phenol-iron complexes/nanoparticles were formed at pH 6.     

Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with beta-carotene

The 4'-hydroxyl group of puerarin, a C-glycoside of the isoflavonoid daidzein, was shown, using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation and stopped-flow spectroscopy and by comparison with the 7-propylpuerarin (A ring derivative) and 4'-propylpuerarin (B ring derivative), to be a more efficient radical scavenger as compared to the 7-hydroxyl group by a factor of 2, a difference increasing upon deprotonation. The difference in radical scavenging agreed with the oxidation potentials (cyclic voltammetry in acetonitrile, 0.1 M Bu4NBF4 at 25 degrees C): E/mV=862+/-3 for puerarin, 905+/-10 for 7-propylpuerarin, and 1064+/-2 for 4'-propylpuerarin relative to ferrocene/ferricenium. In aqueous solution, the reduction potential was shown to decrease for increasing pH, and deprotonation of the 4'-hydroxyl group increased radical scavenging more than deprotonation of the 7-hydroxyl group. The 7-hydroxyl was found to be more acidic (pKa1=7.20+/-0.01 in puerarin and pKa=7.23+/-0.01 in 4'-propylpuerarin) than the 4'-hydroxyl group (pKa2=9.84+/-0.08 in puerarin and pKa=9.51+/-0.02 in 7-propylpuerarin); aqueous solution, ionic strength of 0.1, and 25 degrees C. In phosphatidyl choline liposome of pH 7.4, puerarin and beta-carotene each showed a modest antioxidant activity measured as prolongation of the lag phase for formation of conjugate dienes and using the water-soluble radical initiator APPH with effects of puerarin and beta-carotene being additive. For the lipophilic initiator AMVN, the antioxidative effect decreased for puerarin and increased for beta-carotene as compared to APPH and showed a clear synergism. A regeneration of beta-carotene, effective in the liposome lipid phase as antioxidant, from the cation radical by deprotonated forms of puerarin was demonstrated in 9:1 chloroform/methanol using laser flash photolysis with k2=2.7x10(4) L mol-1 s-1 for the bimolecular process between the cation radical and the puerarin dianion.     

Wavelength dependence of light-induced lipid oxidation and naturally occurring photosensitizers in cheese

Degradation of the potential photosensitizers, riboflavin, chlorophyll, and porphyrin, in Danbo cheese by monochromatic light of wavelength 366, 436, or 546 nm was studied. Three cheeses were investigated, two conventional (16% fat and 25% fat) and one "organic" (25% fat). The effect of illumination was measured by fluorescence spectroscopy and analyzed using multiway and multivariate data analysis. Riboflavin was found to degrade only by 436 nm light, whereas chlorophylls and porphyrins also were influenced by 436 and 546 nm light. The organic cheese had the largest chlorophyll content both before and after similar light exposure, and no change in chlorophyll of this cheese was observed for any of the illumination wavelengths. Upon light exposure of the cheeses, volatile compounds were formed, as analyzed by gas chromatography-mass spectrometry (GC-MS). The relative concentrations of methyl butanoate, 1-pentanol, benzaldehyde, 2-butanone, 2-heptanone, and butyl acetate were found to weakly correlate with the surface fluorescence intensity. 1-Pentanol and the ketones are secondary lipid oxidation products, consistent with a chemical coupling between photosensitizer degradation and formation of volatile lipid oxidation products.     

Role of Gene Interactions in Hybrid Speciation: Evidence from Ancient and Experimental Hybrids

The origin of a new diploid species by means of hybridization requires the successful merger of differentiated parental species' genomes. To study this process, the genomic composition of three experimentally synthesized hybrid lineages was compared with that of an ancient hybrid species. The genomic composition of the synthesized and ancient hybrids was concordant (rs = 0.68, P < 0.0001), indicating that selection to a large extent governs hybrid species formation. Further, nonrandom rates of introgression and significant associations among unlinked markers in each of the three synthesized hybrid lineages imply that interactions between coadapted parental species' genes constrain the genomic composition of hybrid species.