Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish muscle

Variants of sperm whale myoglobin (Mb) were used to assess the mechanism of heme protein-mediated lipid oxidation in washed cod muscle. A myoglobin variant with high hemin affinity (V68T) was an exceptionally poor promoter of lipid oxidation, while a Mb variant with low hemin affinity (H97A) was a potent promoter of lipid oxidation. V68T releases hemin slowly due to the ability of threonine to hydrogen bond with coordinated water and the distal histidine within the heme crevice. H97A rapidly releases hemin because the relatively small alanine residue creates a channel for water to easily enter the heme crevice which weakens the covalent linkage of hemin to the proximal histidine. A variant sensitive to heme degradation (L29F/H64Q) was a weaker promoter of lipid oxidation compared to wild-type Mb. This suggests that degrading the heme ring and releasing iron decreased the ability of Mb to promote lipid oxidation. Free radicals resulting from hemin-mediated decomposition of lipid hydroperoxides have the capacity to propagate lipid oxidation and degrade hemin catalyst. This may explain why heme proteins behave as reactants rather than "catalysts" of lipid oxidation in washed cod. Collectively these studies strongly suggest that released hemin is the critical entity that drives heme protein-mediated lipid oxidation in washed fish muscle.     

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.     

Site e14 in hemoglobins and myoglobins: a key residue that affects hemin loss and lipid oxidation capacity

Fish hemoglobins (Hbs) frequently contain glycine at site E14 while mammalian Hbs contain larger residues (e.g., alanine and serine). These differences were examined by creating structural variants at E14 using recombinant bovine myoglobin (Mb) as a model heme protein that contains alanine at E14. The Ala(E14)Gly mutation increased k(ox) and hemin loss 3-fold and 45-fold, respectively. Glycine at E14 creates a channel for solvent to enter the heme crevice, which enhances autoxidation and hemin loss rates. Hydration of the proximal heme pocket facilitates hemin loss because protonation of the proximal histidine weakens the linkage of the imidazole group to the iron atom of the hemin moiety. Ala(E14)Gly promoted lipid oxidation in washed fish muscle more rapidly during iced storage compared to wild type Mb at pH 5.7. This suggested that the rapid hemin loss from Ala(E14)Gly accelerated lipid oxidation. Ala(E14)Ser and Ala(E14)Val had little effect on k(ox) but somewhat accelerated net hemin loss. These studies suggest that enhanced access of solvent to the heme crevice of many fish Hbs at site E14 facilitates rapid hemin loss and moderately accelerates autoxidation. This likely is part of the reason fish Hbs promote lipid oxidation much more effectively compared to mammalian Hbs.     

Ferrochelatase catalyzes the formation of Zn-protoporphyrin of dry-cured ham via the conversion reaction from heme in meat

Ferrochelatase (FECH), the enzyme at the last step of the heme-biosynthetic pathway, is involved in the formation of Zn-protoporphyrin via an iron-removal reaction of heme. To improve the efficacy of the formation of Zn-protoporphyrin from heme, the use of recombinant FECHs from porcine, yeast, and bacteria was examined. Incubation of FECH with myoglobin in the presence of ascorbic acid and cysteine resulted in the efficient conversion of myoglobin-heme to Zn-protoporphyrin. Exogenously added recombinant yeast FECH facilitates the production of Zn-protoporphyrin from myoglobin-heme and heme in meat, via the replacement of iron in the protoporphyrin ring by zinc ions. A large amount of Zn-protoporphyrin was also generated by the catalysis of FECH using an intact piece of meat as a substrate. These findings can open up possible approaches for the generation of a nontoxic bright pigment, Zn-protoporphyrin, to shorten the incubation time required to produce dry-cured ham.     

Perfluorodecanoic acid binding to hemoproteins: new insights from spectroscopic studies

Perfluorodecanoic acid (PFDA), a representative of the perfluoroalkyl acids, poses a great threat to humans and animals via food and other potential sources. In this work, we determined the effects of PFDA binding to two hemoproteins, bovine hemoglobin (BHb) and myoglobin (Mb). Using fluorescence spectroscopy, we found that PFDA greatly enhanced the fluorescence intensity of both hemoproteins, while perfluorooctanoic acid (PFOA) and perfluoropentanoic acid (PFPA) have minimal effects on the fluorescence. UV-vis absorption (UV) spectroscopy showed that PFDA induced the unfolding of the hemoproteins accompanied by exposure of the heme pocket and facilitating the formation of hemichrome. Additionally, as shown by the circular dichroism (CD) data, PFDA altered the secondary structure of both BHb and Mb. This work elucidates the interaction mechanism of PFDA with two hemoproteins.     

Mechanisms of heme protein-mediated lipid oxidation using hemoglobin and myoglobin variants in raw and heated washed muscle

The hemoglobin variant rHb 0.1, which possesses a decreased ability to form subunits, stimulated lipid oxidation in washed fish muscle less effectively as compared to wild-type hemoglobin (rHb 0.0). This could be due to the lower hemin affinity and more rapid autoxidation rate of subunits as compared to tetramers. To differentiate between hemin affinity and autoxidation effects, ferrous V68T Mb was compared to ferrous wild-type myoglobin (WT Mb). WT Mb has a more rapid hemin loss rate (25-fold) than does V68T, while V68T autoxidized more rapidly than did WT Mb (60-fold). Ferrous WT Mb promoted TBARS and lipid peroxide formation more rapidly than did ferrous V68T (p < 0.01). This indicated hemin loss rate was more critical in determining onset of lipid oxidation as compared to autoxidation rate. Hemin alone was capable of stimulating lipid oxidation. Albumin enhanced the ability of hemin to promote lipid oxidation. MetMb promoted lipid oxidation more effectively than did ferrous Mb, which could be due to the lower hemin affinity of metMb as compared to that of ferrous Mb. EDTA, an iron chelator, had no effect on the rate or extent of lipid oxidation mediated by Mb in the cooked system. Variants with a 975-fold range of hemin affinities promoted lipid oxidation with equivalent efficacy in cooked washed cod contrary to results in uncooked washed cod. The cooking temperatures apparently denature the globin and release hemin reactant to such an extent that the impact of hemin affinity on lipid oxidation observed in the raw state is negated in the cooked state. These studies collectively suggest released hemin is of primary importance in promoting lipid oxidation in raw and cooked washed fish muscle.     

Pro-oxidative characteristics of trout hemoglobin and myoglobin: a role for released heme in oxidation of lipids

The molecular mass of trout myoglobin was 16017 Da based on electrospray ionization mass spectrometry. A Root effect (low oxygen affinity at pH 6.3) was determined in trout hemoglobin but not myoglobin. At pH 6.3, myoglobin autoxidized more rapidly (3.5-fold) as compared to anodic hemoglobin. Anodic hemoglobin was a better catalyst of lipid oxidation in washed cod muscle as compared to myoglobin at pH 6.3. This suggested that some process other than met heme protein formation was the rate-limiting step in lipid oxidation processes. Heme loss rates were determined using the apomyoglobin mutant H64Y prepared from sperm whale. Anodic hemoglobin released its heme group much more rapidly than myoglobin. In comparisons of anodic and cathodic hemoglobins, heme loss rate better predicted the onset of lipid oxidation than autoxidation rate. These studies collectively suggest that heme dissociation has a primary role in the ability of different heme proteins to promote lipid oxidation processes.     

Effects of storage atmosphere and heme state on the color and visible reflectance spectra of salmon ( Salmo salar ) fillets

It has previously been observed that the color of mackerel muscle is dependent on the status of heme as myoglobin and hemoglobin and hence the storage atmosphere. This study gives strong indications of this being the case also in salmon. Three different storage conditions were used to promote the oxidized, reduced, and carbon monoxide (CO) bound forms of heme in salmon and mackerel fillets. Color determination (instrumental color analysis, imaging, and sensory evaluation) and spectroscopic measurements were performed to study how spectral changes corresponded to color variations. Storage in CO significantly increased the redness in mackerel. This was also seen in salmon to such a degree that it was visible over normal levels of salmon carotenoids. Air storage increased the yellowness and reduced the redness in mackerel, but this effect was partly concealed in salmon by the astaxanthin absorption. The spectral differences due to storage condition could be ascribed to the spectral features characterizing heme of different oxidation states and bound to different ligands. The status of heme should therefore always be considered when experiments related to salmon color are performed. The findings could help in the understanding, control, and prediction of color loss in salmon during processing, storage, and transport.     

Reactivation of broccoli peroxidases: structural changes of partially denatured isoenzymes

Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into an inactive structural species, similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase, as well as molecular rearrangement of the structure around the heme, occurs during incubation at approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed. Keywords: Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.     

Potential of peroxynitrite to alter the color of myoglobin in muscle foods

Superoxide anion and nitric oxide can react to form the highly oxidizing species peroxynitrite. The objective of this research was to determine if peroxynitrite can promote the discoloration of myoglobin under conditions expected in muscle foods. Reagent peroxynitrite (25-100 microM) caused rapid and extensive formation of metmyoglobin from oxymyoglobin with the majority of metmyoglobin formation occurring during the first 5-10 min of incubation. Carbon dioxide caused a small decrease in the ability of peroxynitrite to oxidize oxymyoglobin, and peroxynitrite-promoted conversion of oxymyoglobin to metmyoglobin increased with decreasing pH (5.5-7.0). Differential scanning calorimetry suggested that peroxynitrite caused minimal changes in myoglobin structure. These results indicate that peroxynitrite can promote the conversion of oxymyoglobin to metmyoglobin under the conditions expected in muscle foods.     

Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study

This study was designed to assess the interactions of heme with peptides produced by enzyme hydrolysis of hemoglobin, and their relationship with heme iron absorption. Bovine hemoglobin was hydrolyzed by pepsin or by subtilisin, which differ in their hydrolysis processes. The hydrolysis rate ranged from 0 (native hemoglobin) to 15%. Heme solubility and heme-peptides interactions were compared to iron absorption by the Ussing chamber model, at intestinal pH (7.5). Increasing hemoglobin hydrolysis enhanced iron absorption; the highest value was reached between 8 and 11% hydrolysis, whatever the enzyme used. Comparing the products of hydrolysis of the two enzymes showed that heme iron absorption depends not only on its solubility, but relies mainly on the balance between the strength of heme-peptides and the polymerization rate of heme.     

New compounds obtained by evolution and oxidation of malvidin 3-O-glucoside in ethanolic medium

Two new colorless phenolic compounds were formed from malvidin 3- O-glucoside incubated in an ethanolic solution. Their structures were characterized by means of one- and two-dimensional NMR analysis and through electrospray ionization-mass spectrometry. As compared to the structure of the initial anthocyanin skeleton, the first new compound showed the presence of two fused five-membered rings replacing the pyran ring and of a carbonyl function on the 2-position. The first five-membered ring was shown to result from the formation of a new linkage between the B ring 6'-position and the C ring 4-position, while the second was a dihydro furan ring with an oxygenated ether linkage between the 8a-position and the 3-position. The second isolated compound was shown to have similar structure with an ethyl ether moiety in the 3-position instead of the glucose moiety. A mechanism explaining the formation of the isolated compounds involving the passage through the chalcone form of the anthocyanin and an oxidation process is proposed.     

Effect of residual ascorbate on determination of nitrite in commercial cured meat products

Residual ascorbate in cured meat slurries results in different amounts of pigment being produced from different Griess reagent combinations. The phenomenon was used to study residual ascorbate in commercial cured meat products which had a variety of textures, acidities, moisture and meat content, fat, homogeneity, initial nitrite, and processing conditions. Diluting and heating the samples according to the AOAC procedure did not completely eliminate the ascorbate interference, but making the sample alkaline did. Determining nitrite separately in supernate and precipitate from the first dilution showed the effect of heating to be the elimination of interferences and solubilization or extraction of nitrite from the precipitate.     

Distinctive antioxidant and antiinflammatory effects of flavonols

The antioxidant and antiinflammatory effects of flavonols have been suggested to be structure-related. Results revealed that selected flavonols, including fisetin (F), kaempferol (K), morin (MO), myricetin (MY), and quercetin (Q), exhibited distinctive free radical scavenging properties against different kinds of free radicals. The H donation (DPPH bleaching) potential was Q > F approximately equals MY > MO > K, indicating that the presence of a 3',4'-catechol moiety in the B ring correlated with high activity. The 4'-OH in the B ring was suggested to be important for reducing xanthing/xanthine oxidase-generated superoxide; while an additional OH moiety on the ortho sites (3' or 5') attenuated the effect as the observed inhibitory potency was K approximately equals MO > Q > F > MY. The relative inhibitory effect for Fenton-mediated hydroxyl radical was K approximately equals MO approximately equals Q > F > MY. This result implies the involvement of 4-keto, 5-OH region in Fe++ chelating and the negative effect of pyrogallol moiety in the B ring. Similar to the inhibitory activity against a N-formyl-methionyl-leucyl-phenylalanine (f-MLP)-stimulated oxidative burst in human polymorphonuclear neutrophils (PMN), our result showed that the structural peculiarity of the di-OH in the B ring obviously rendered F, Q, and MO more potent as ROS inhibitors than MY and K, which have tri- and mono-OH in the B ring, respectively. All of the previous data indicated that the structure prerequisite to reinforce the free radical scavenging activity varies with the type of free radical. We further analyzed the effects of flavonols on nitric oxide (NO) production in endotoxin-stimulated murine macrophages, RAW264.7 cells. Results showed that all flavonols (up to 10 microM) inhibited NO production without exerting detectable cytotoxicity. F, K, and Q dose-dependently repressed iNOS mRNA expression and prostaglandin E2 (PGE2) production, in part through an attenuating NF-kappaB signaling pathway. This result indicates that flavonols, despite structural similarity, have different antioxidant and antiinflammatory effects.     

Development of an ELISA for the detection of the residues of the fungicide iprovalicarb

A competitive indirect enzyme-linked immunosorbent assay was developed for the fungicide iprovalicarb, using a polyclonal antibody produced against a hapten conjugated through the carboxyl group on the benzene ring to keyhole limpet hemocyanin. Under an optimized condition using a heterologous format, an IC(50) of 3.51 ng/mL and the lowest detection limit of 0.065 ng/mL were obtained. When the isopropoxy group was removed from the iprovalicarb structure for the synthesis of a hapten, the resulting hapten was not successful as an immunogen, indicating that the isopropyl moiety was an important epitope, as evidenced by the cross-reactivities of some structurally related compounds. When applied to the real crop and water samples, the recoveries were in the range of 80.52-144.70% (n = 4) and 72.11-100.43% (n = 4), respectively. Accordingly, this ELISA can be used as a useful method for monitoring iprovalicarb residues in crop and water samples.     

不同干燥方式对牛肉干物性特性的影响

为了探究中红外-热风组合（combined mid-infrared and hot air,CMIHA）干燥对牛肉干物性特性的影响,该文研究了CMIHA干燥工艺和热风（hot air,HA）干燥工艺对牛肉干干燥过程中色泽与质构特性的影响。通过分析牛肉干在2种干燥过程中亮度值、红度值、黄度值的变化,结合肌红蛋白、高铁肌红蛋白、氧合肌红蛋白及血红素铁的含量变化,研究干燥工艺对牛肉干色泽的影响;通过测定干燥过程中牛肉干嫩度和质构分析（texture profile analysis,TPA）的变化,并基于扫描电镜（scanning electron microscopy,SEM）、透射电镜（transmission electron microscope,TEM）以及核磁成像系统（magnetic resonance imaging,MRI）研究干燥工艺对牛肉干微观结构及氢质子密度的影响,分析干燥工艺对牛肉干质构的影响。结果表明：与HA干燥相比,CMIHA干燥后期能够显著（P<0.05）改善牛肉干的色泽,提高牛肉干的嫩度,增加牛肉干的弹性和咀嚼性;另外,与HA干燥相比,CMIHA干燥能够降低肌红蛋白的氧化,增加氧合肌红蛋白、肌红蛋白和血红素铁的含量,赋予牛肉干较好的色泽;SEM与TEM观察结果表明,牛肉在干燥过程中,肌肉的微观结构均发生一定的收缩,但与HA干燥相比,CMIHA干燥的牛肉干肌肉微观结构收缩程度较轻,同时微观结构保持较好;MRI观察结果表明,与HA干燥相比,牛肉干在CMIHA干燥过程中,内外水分分布较均匀,且收缩程度较轻。与生产中常规使用的HA干燥相比,CMIHA干燥能够降低肌红蛋白的氧化和肌肉微观结构的收缩,增加内外水分子分布的均一性,从而改善牛肉干的色泽和质构,提高牛肉干的物性特性。研究结果为CMIHA干燥在牛肉干方面的应用提供理论依据与技术参考。     

Concentration effects in myoglobin-catalyzed peroxidation of linoleate

The concentration of the free fatty acid anion linoleate was found to be important for the pro-oxidative activity of metmyoglobin, MbFe(III), and for mixtures of metmyoglobin and hydrogen peroxide, MbFe(III)/H(2)O(2), to yield perferrylmyoglobin, (*)MbFe(IV)=O, whereas for ferrylmyoglobin, MbFe(IV)=O, no concentration effect was noted as studied in linoleate emulsions (pH 7.4 and 25 degrees C). Determination of conjugated dienes using second-derivative absorption spectroscopy, changes in Soret band absorbance, and spin-trapping ESR spectroscopy with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) as the spin trap were used to evaluate the pro-oxidative activity of myoglobins. At a linoleate (LA)/heme protein (HP) ratio of 100, no MbFe(III)-induced linoleate peroxidation was observed, as MbFe(III) was converted to its non-pro-oxidative low-spin derivative, hemichrome, independently of the presence of H(2)O(2). At higher LA/HP ratios, linoleate peroxidation was initiated by the addition of MbFe(III), both in the presence and in the absence of H(2)O(2). This proceeded with denaturation of MbFe(III), as followed by changes in Soret absorption band, which most probably release or expose the heme group to the environment and thereby permit hematin-induced lipid peroxidation. The obtained results show that the mechanism by which MbFe(IV)=O initiates linoleate peroxidation is different from MbFe(III)- and MbFe(III)/H(2)O(2)-initiated linoleate peroxidation. The shift in mechanism between heme protein cleavage of lipid hydroperoxides and hematin-induced lipid peroxidation is discussed in relation to oxidative progress in biological systems and muscle-based foods.     

基于稳态空间分辨光谱的猪肉肌红蛋白检测方法

The objective of this work was to determine the concentration of myoglobin in fresh pork meat and establish a fast non-invasive method using steady spatially-resolved spectroscopy (SRS). The concentration of myoglobin was calculated based on absorbance coefficients at 640, 760, 800, 850 nm. The results showed that the value of myoglobin using SRS was significantly correlated (r2=0.955) to traditional methods with the accuracy of 3.6%F.S. The results showed that the concentration of myoglobin can be measured by using near infrared steady spatially-resolved spectroscopy.     

板栗壳色素的提取、纯化及稳定性（简报）

该文以板栗壳为材料,研究板栗壳色素的提取、纯化工艺及纯化色素的稳定性,为板栗壳色素的推广应用提供理论基础和实验数据.结果表明,板栗壳色素易溶于碱性水溶液,不溶或难溶于非极性溶剂.用1%的NaOH提取色素,石油醚和乙酸乙酯分别笨取3次后获得粗提色素,粗提色素经醇沉、酸沉及霞结晶法三种方法纯化后通过薄层层析证明色素纯度较高.紫外-可见光谱分析表明,纯化色素的0.01%水溶液在223 nm和264 nm处有明显吸收峰,0.01%甲醇溶液在218 m和264 nm处有明显吸收峰,推测板栗壳色素含有苯环及酚羟基.纯化色素的稳定性试验表明,色素264.nm处吸光度对pH值较敏感.510 nm处吸光度在pH为2.0至6.0时.呈逐渐增加的趋势,在pH为8.0至14.0时变化不大,pH=10.0条件下测定510 nm处吸光度可以作为色素定量的方法;不同金属离子对色素有不同影响;色素对光、热、氧化剂及还原剂的耐受力较强.     

Oxidation of quercetin by salivary components. Quercetin-dependent reduction of salivary nitrite under acidic conditions producing nitric oxide

Under acidic conditions, nitrite is protonated to nitrous acid (pK(a) = 3.2-3.4) that can be transformed into nitric oxide by self-decomposition and reduction. When sodium nitrite was mixed with quercetin at pH 1-2, quercetin was oxidized producing nitric oxide. In addition to quercetin, kaempferol and quercetin 4'-glucoside were also oxidized by nitrous acid, but oxidation of apigenin, luteolin, and rutin was slow compared to oxidation of the above flavonols. These results suggested that flavonols, which have a free hydroxyl group at carbon position 3, can readily reduce nitrous acid to nitric oxide. When the pH of saliva was decreased to 1-2, formation of nitric oxide was observed. The nitric oxide formation was enhanced by quercetin, and during this process quercetin was oxidized. These results indicate that there is a possibility of reactions between phenolics and nitrous acid derived from salivary nitrite in the stomach.