Ferric Myoglobin Research Articles

Photoexcitation dynamics of azide ion bound ferric myoglobin probed by femtosecond infrared spectroscopy

AbstractThe photoexcitation dynamics of N3−‐bound ferric myoglobin (MbN3) were investigated after exciting MbN3 in D2O at 283 K with a 575 nm pulse by probing the anti‐symmetric stretching mode of the azide. Global fitting of the overall time‐resolved spectra revealed that thermal relaxation of two stretching bands proceeded with a time constant of 6 ps, and that a new absorption band formed and decayed with time constants of 0.6 and 23 ps, respectively. The new absorption near 2040 cm−1 was attributed to the high‐spin species 2.4 kJ/mol above the low‐spin species, as the excited low‐spin relaxes thermally via the high‐spin species. However, this absorption could also arise from deligated N3̄ remaining within the protein. The decay of this absorption can be interpreted as either spin transition of the high‐spin species into the low‐spin species or geminate recombination of the dissociated N3̄. The implications of both interpretations are discussed.

Dioxygen and glucose force motion of the electron-transfer switch in the iron(III) flavohemoglobin-type nitric oxide dioxygenase

Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O2 complex formation and O2-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O2 complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O2 complex and O2-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O2 complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O2 on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common ‘oxy-met’ state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in flavohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O2 storage functions. The results support the proposed roles of a ferric O2 intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.

Myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI

Crush syndrome (CS) is a life-threatening illness in traffic accidents and earthquakes. Crush syndrome-induced acute kidney injury (CS-AKI) is considered to be mainly due to myoglobin (Mb) circulation and deposition after skeletal muscle ruptures and releases. Macrophages are the primary immune cells that fight foreign substances and play critical roles in regulating the body’s natural immune response. However, what effect does myoglobin have on macrophages and the mechanisms involved in the CS-AKI remain unclear. This study aims to look into how myoglobin affects macrophages of the CS-AKI model. C57BL/6 mice were used to construct the CS-AKI model by digital crush platform. Biochemical analysis and renal histology confirmed the successful establishment of the CS-AKI mouse model. Ferrous myoglobin was used to treat Raw264.7 macrophages to mimic the CS-AKI cell model in vitro. The macrophage polarization toward M1 type and activation of RIG-I as myoglobin sensor were verified by real-time quantitative PCR (qPCR), Western blotting (WB), and immunofluorescence (IF). Macrophage pyroptosis was observed under light microscopy. The interaction between RIG-I and caspase1 was subsequently explored by co-immunoprecipitation (Co-IP) and IF. Small interfering RNA (siRIG-I) and pyroptosis inhibitor dimethyl fumarate (DMF) were used to verify the role of macrophage polarization and pyroptosis in CS-AKI. In the kidney tissue of CS-AKI mice, macrophage infiltration and M1 type were found. We also detected that in the cell model of CS-AKI in vitro, ferrous myoglobin treatment promoted macrophages polarization to M1. Meanwhile, we observed pyroptosis, and myoglobin activated the RIG-I/Caspase1/GSDMD signaling pathway. In addition, pyroptosis inhibitor DMF not only alleviated kidney injury of CS-AKI mice but also inhibited macrophage polarization to M1 phenotype and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway. Our research found that myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI.

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Due to fast relaxation processes of transition metal ions, electron paramagnetic resonance (EPR) spectroscopy of metalloproteins needs to be performed at cryogenic temperatures. To avoid damaging the biological system upon freezing, a cryoprotectant is generally added to the sample as a glassing agent. Even though cryoprotectants are expected to be inert substances, evidences in literature show their non-innocent role in altering the shape of EPR spectra of proteins and biological objects in general. In this work we conduct a systematic study on the impact of several experimental factors—such as buffer composition, choice of cryoprotectant, pH and temperature—on the EPR spectrum of myoglobin, taken as a reference system for being a well-characterized heme-containing protein. We focus on high-pH buffers to induce and investigate the alkaline transition of ferric myoglobin (pKa ~ 8.9). A combined approach of continuous-wave EPR and UV–visible absorption spectroscopy shows that using particular pairs of buffers and cryoprotectants determines a considerable pH variation in the sample and that this effect is enhanced at cryogenic temperature. In addition, phase memory times were measured to evaluate the efficiency of different cryoprotectants and compared with spectral linewidths in continuous-wave EPR. Our findings suggest that among the selected cryoprotectants ethylene glycol is rather effective, even more than the widely used glycerol, without having unwanted effects.

RIG-I, a novel DAMPs sensor for myoglobin, activates NF-\u03baB/caspase-3 signaling in CS-AKI model

BackgroundAcute kidney injury (AKI) is the main life-threatening complication of crush syndrome (CS), and myoglobin is accepted as the main pathogenic factor. The pattern recognition receptor retinoicacid-inducible gene I (RIG-I) has been reported to exert anti-viral effects function in the innate immune response. However, it is not clear whether RIG-I plays a role in CS-AKI. The present research was carried out to explore the role of RIG-I in CS-AKI.MethodsSprague-Dawley rats were randomly divided into two groups: the sham and CS groups (n = 12). After administration of anesthesia, the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions. The rats in both groups were denied access to food and water. Rats were sacrificed at 12 h or 36 h after pressure was relieved. The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination. In addition, RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI. Furthermore, NRK-52E cells were treated with 200 μmol/L ferrous myoglobin to mimic CS-AKI at the cellular level. The cells and cell supernatant samples were collected at 6 h or 24 h. Small interfering RNAs (siRNA) was used to knock down RIG-I expression. The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative Real-time PCR (qPCR), Western blotting analysis, and immunohistochemistry (IHC) staining. Tumor necrosis factor-α (TNF-α) was detected by ELISA. Co-Immunoprecipitation (Co-IP) assays were used to detect the interaction between RIG-I and myoglobin.ResultsRNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway. qPCR, Western blotting, and IHC assays showed that RIG-I, nuclear factor kappa-B (NF-κB) P65, p-P65, and the apoptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group (P < 0.05). However, the levels of interferon regulatory factor 3 (IRF3), p-IRF3 and the antiviral factor interferon-beta (IFN-β) showed no significant changes between the sham and CS groups. Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group. Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis.ConclusionRIG-I is a novel damage-associated molecular patterns (DAMPs) sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI. In the development of CS-AKI, specific intervention in the RIG-I pathway might be a potential therapeutic strategy for CS-AKI.

Activation of aryl hydrocarbon receptor by 6-formylindolo[3,2-b]carbazole alleviated acute kidney injury by repressing inflammation and apoptosis.

Acute kidney injury (AKI) is a multifactorial disease of various aetiologies. Aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor that responds to ligands to induce or repress gene expressions, thereby regulating a diverse spectrum of biological or pathophysiologic effects. However, the effect of AhR on AKI remains unknown. A single intraperitoneal injection of 50% glycerol was performed to induce rhabdomyolysis in C57BL/6J mice. The bilateral renal pedicles were occluded for 30 minutes and then removed to stimulate renal I/R injury. 6‐formylindolo[3,2‐b]carbazole (FICZ), a photo‐oxidation product of tryptophan with a high affinity for AhR, was used. The in vitro study was performed on HK‐2 cells. Ferrous myoglobin and FICZ was dissolved in the medium in different cell groups. Treatment with AhR agonist FICZ significantly alleviated the elevation of serum creatinine and urea in AKI. AKI modelling‐induced renal damage was attenuated by FICZ. AhR mainly expressed in proximal tubular cells and could be activated by FICZ administration. Meanwhile, AKI triggered the production of pro‐inflammatory cytokines in injured kidneys, while FICZ inhibited their expressions. Furthermore, FICZ effectively reversed cell apoptosis in AKI models. Mechanistically, AKI stimulated the activation of NF‐κB and JNK pathways in the kidneys, while FICZ significantly suppressed these corresponding protein expressions. For the in vitro study, FICZ also inhibited inflammation and apoptosis in myoglobin or H/R‐stimulated HK‐2 cells. In summary, agonism of AhR by FICZ alleviated rhabdomyolysis and I/R‐induced AKI. FICZ inhibited inflammation and apoptosis via suppressing NF‐κB and JNK pathways in proximal tubular cells.

Rhabdomyolysis-Induced AKI Was Ameliorated in NLRP3 KO Mice via Alleviation of Mitochondrial Lipid Peroxidation in Renal Tubular Cells.

Introduction: A recent study showed that early renal tubular injury is ameliorated in Nod-like receptor pyrin domain-containing protein 3 (NLRP3) KO mice with rhabdomyolysis-induced acute kidney injury (RIAKI). However, the precise mechanism has not been determined. Therefore, we investigated the role of NLRP3 in renal tubular cells in RIAKI. Methods: Glycerol-mediated RIAKI was induced in NLRP3 KO and wild-type (WT) mice. The mice were euthanized 24 h after glycerol injection, and both kidneys and plasma were collected. HKC-8 cells were treated with ferrous myoglobin to mimic a rhabdomyolytic environment. Results: Glycerol injection led to increase serum creatinine, aspartate aminotransferase (AST), and renal kidney injury molecule-1 (KIM-1) level; renal tubular necrosis; and apoptosis. Renal injury was attenuated in NLRP3 KO mice, while muscle damage and renal neutrophil recruitment did not differ between NLRP3 KO mice and WT mice. Following glycerin injection, increases in cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), and a decrease in the glutathione peroxidase 4 (GPX-4) level were observed in the kidneys of mice with RIAKI, and these changes were alleviated in the kidneys of NLRP3 KO mice. NLRP3 was upregulated, and cell viability was suppressed in HKC-8 cells treated with ferrous myoglobin. Myoglobin-induced apoptosis and lipid peroxidation were significantly decreased in siNLRP3-treated HKC-8 cells compared to ferrous myoglobin-treated HKC-8 cells. Myoglobin reduced the mitochondrial membrane potential and increased mitochondrial fission and reactive oxygen species (ROS) and lipid peroxidation levels, which were restored to normal levels in NLRP3-depleted HKC-8 cells. Conclusions: NLRP3 depletion ameliorated renal tubular injury in a murine glycerol-induced acute kidney injury (AKI) model. A lack of NLRP3 improved tubular cell viability via attenuation of myoglobin-induced mitochondrial injury and lipid peroxidation, which might be the critical factor in protecting the kidney.

Fluorescence spectra of nutrients in chicken and skin under baking conditions

The chicken and skin were studied by fluorescence spectroscopy. The three-dimensional fluorescence spectra of chicken and skin were measured, It was concluded that the optimal excitation wavelength corresponding to the fluorescence peak of 330 nm in chicken was 270 nm, and that of 475 nm and 520 nm was 370 nm. The optimal excitation wavelength corresponding to the 475 nm fluorescence peak of chicken skin was 370 nm. The fluorescence spectra of chicken skin and chicken excited by the wavelength of 370 nm were analyzed. The fluorescence peak came from myoglobin at 555 nm at the beginning of roasting, and the contribution of fluorescence peak came from oxygenated myoglobin at 540 nm and 580 nm as the baking time increased. When the baking time was more than 12 min, the fluorescence intensity of oxygenated myoglobin at 580 nm was close to zero. At this point, a considerable part of myoglobin had been converted into ferric myoglobin, while Fe3+ in ferric myoglobin could not be absorbed by human body. At this point, the skin of chicken was no longer suitable for large amounts of consumption, while the relative performance of chicken was not obvious.

The reactions of hydropersulfides (RSSH) with myoglobin

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

Immunomodulatory role of recombinant human erythropoietin in acute kidney injury induced by crush syndrome via inhibition of the TLR4/NF-κB signaling pathway in macrophages

Objective: The present study aimed to investigate whether recombinant human erythropoietin (rHuEPO) plays an immunomodulatory function by regulating the TLR4/NF-κB signaling pathway.Materials and methods: C57BL/6 mice were intraperitoneally injected with rHuEPO and, half an hour later, with 50% glycerol at the dose of 7.5 ml/kg to induce crush syndrome (CS)-acute kidney injury (AKI). The levels of TNF-α, IL-1β, IL-6, serum creatinine (Scr), and creatine kinase (CK) were measured. The kidney tissues were analyzed by HE staining, and macrophage infiltration was detected by immunohistochemistry. Double immunofluorescence staining, RT-qPCR, and western blotting were conducted to analyze TLR4/NF-κB p65 expression. Ferrous myoglobin was co-cultured with RAW264.7 cells to mimic crush injury and the production of proinflammatory cytokines. The expression levels of TLR4 and NF-κB p65 were measured.Results: In vivo study results revealed that rHuEPO ameliorated renal function, tissue damage, production of proinflammatory cytokines, and macrophage infiltration in the kidneys. The protein and mRNA expression levels of genes involved in the TLR4/NF-κB signaling pathway in CS-induced AKI mice were upregulated (p < .05). Meanwhile, the expression levels of TLR4, NF-κB p65, and proinflammatory cytokines in RAW264.7 cells were downregulated in CS-AKI mice injected with rHuEPO (p < .05).Conclusions: Our results demonstrated the immunomodulatory capacity of rHuEPO and confirmed that rHuEPO exerts protective effects against CS-induced AKI by regulating the TLR4/NF-κB signaling pathway in macrophages. Therefore, our findings highlight the therapeutic potential of rHuEPO in improving the prognosis of CS-AKI patients.

Interrelationship among ferrous myoglobin, lipid and protein oxidations in rabbit meat during refrigerated and superchilled storage

This work investigated the interrelationship among myoglobin, lipid, and protein oxidations in rabbit meat during refrigerated and superchilled storage. Peroxide value gradually increased (p < 0.05) and decreased thereafter (p > 0.05) with increased storage time for both storage treatments. Thiobarbituric acid-reactive substances (TBARS) significantly increased (p < 0.05) throughout the storage time for the storage temperature considered. The metmyoglobin proportion markedly increased (p < 0.05) and remained constant thereafter (p > 0.05). For both storage temperature levels, the extractable heme iron content significantly declined (p < 0.05), whereas non-heme iron content considerably increased (p < 0.05). With prolonged storage time, the protein carbonyl content significantly increased (p < 0.05), along with significant loss in sulfhydryl content (p < 0.05). Superchilled samples maintained lower rates of increase in metmyoglobin content, TBARS, and protein carbonyl content compared with refrigerated samples. Moreover, principal component analysis presented good correlations between TBARS, protein carbonyls and metmyoglobin content. Taken together, myoglobin, lipid and protein oxidations occurred concurrently in rabbit meat during both storage treatments and each oxidation process seemed to promote the other.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury.

Background:Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway.Methods:C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected.Results:In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P < 0.01). CLI-095 or PDTC administration suppressed proinflammatory cytokine (TNF-α, IL-6, and IL-1β) production and macrophage infiltration into the kidney (P < 0.01). Moreover, in an in vitro study, CLI-095 or PDTC suppressed myoglobin-induced expression of TLR4, NF-κB, and proinflammatory cytokine levels in macrophage RAW264.7 cells (P < 0.01).Conclusion:The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

Mössbauer spectroscopy as a probe of electric field in heme pocket of deoxyheme proteins: theoretical approach

Chemical reactions taking place in active centers of different enzymes are controlled by electric fields created by the protein in these centers. These electric fields can be experimentally detected by different experimental techniques (infrared absorption, NMR, etc.). In this paper, we use quantum chemical calculations to show that Mössbauer spectroscopy can be also used to study protein electric field. We study effect of both the model and protein electric fields on the magnitude of quadrupole splitting of Mössbauer spectra of the high-spin ferrous myoglobin and its models. It is shown that the quadrupole splitting is notably affected by the protein electric field. This result also explains a number of the experimental data.

Improved stopped‐flow time‐resolved resonance Raman spectroscopy device for studying enzymatic reactions

An improved stopped‐flow resonance Raman spectroscopy device was constructed using a stopped‐flow mixer with a dead time of 3 ms and a mixing volume of 0.1 mL. The device was tested using myoglobin, where the formation reaction of a high‐valent heme species, ferryl‐oxo heme, was monitored by time‐resolved resonance Raman spectroscopy after mixing a ferric myoglobin solution with a hydrogen peroxide solution. The ferryl‐oxo heme formation rate constant obtained by Raman spectroscopy is in good agreement with the rate constant obtained by conventional stopped‐flow absorption spectroscopy for the same reaction under the same conditions. It is proved by these results that the present device is generally applicable to enzyme–substrate reactions with a significantly higher time resolution than previously reported. Copyright © 2017 John Wiley &amp; Sons, Ltd.

Biological Membrane-Packed Mesenchymal Stem Cells Treat Acute Kidney Disease by Ameliorating Mitochondrial-Related Apoptosis

The mortality of rhabdomyolysis-induced AKI remains high because no effective therapy exists. We investigated a new therapeutic method using MSCs. The aim of this study was to investigate the therapeutic potential and anti-apoptotic mechanisms of action of MSCs in the treatment of AKI induced by glycerol in vivo and in vitro. We used Duragen as a biological membrane to pack MSCs on the glycerol-injured renal tissue in vivo. The anti-apoptotic mechanism was investigated. In vitro, HK-2 cells were incubated with ferrous myoglobin and MSCs-conditioned medium, followed by cell proliferation and apoptosis assays. We founded that packing MSCs on the injured renal tissue preserved renal function, ameliorated renal tubular lesions, and reduced apoptosis in the mice with glycerol-induced AKI. The MSC-conditioned medium improved HK-2 cell viability and inhibited apoptosis. These effects were reversed by the PI3K inhibitor LY294002. Biological membrane packing of MSCs on the renal tissue has a therapeutic rescue function by inhibiting cell apoptosis in vivo. MSCs protect renal cells from apoptosis induced by myoglobin in vitro. We have thus demonstrated MSCs reduced rhabdomyolysis-associated renal injury and cell apoptosis by activating the PI3K/Akt pathway and inhibiting apoptosis.

Gene Microarray Integrated with High-Throughput Proteomics for the Discovery of Transthyretin in Rhabdomyolysis-Induced Acute Kidney Injury

Background/Aims: Rhabdomyolysis, one of the leading causes of acute kidney injury (AKI), develops after trauma, drug toxicity, infections, burns, and physical exertion. The aim of this study was to investigate differences in gene and protein expression to elucidate the pathogenesis of rhabdomyolysis (RM)-induced AKI. Methods: In this study, we used glycerol induced renal injury as a model of RM-induced AKI. Affymetrix U133 plus 2.0 microarrays were used to perform gene microarray analysis. Isobaric tagging with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS) was applied to screen and identify differentially expressed proteins between RM-induced AKI and normal murine renal tissue. Verification experiments included immunohistochemistry (IHC), real-time PCR, Western blotting, and the measurement of ATP and ROS production. HK-2 cells were incubated in vitro with ferrous myoglobin and pcDNA-TTR, followed by assays to detect cell proliferation, ROS and apoptosis. Results: According to gene microarray and iTRAQ-MS analysis, we screened 17 common elements. After multiple analyses, we selected transthyretin (TTR) as our focus and investigated TTR in the kidney. Verification experiments with IHC confirmed differential expression levels of TTR proteins. Furthermore, Western blotting showed a stepwise decrease in TTR in AKI renal tissues. Cell-based experiments showed that overexpression of TTR could improve HK-2 cell viability and inhibit apoptosis. TTR reduced apoptosis by decreasing the accumulation of reactive oxygen species (ROS). Conclusion: This study reports a possible mechanism for RM-induced AKI and suggests that reductions in TTR could increase the generation of ROS and induce apoptosis. TTR may be a potentially valuable target for RM-induced AKI.

Muscle-Specific Color Stability in Fallow Deer (Dama Dama) Meat

ObjectivesColor stability of fresh meats is the single most important trait affecting the consumers’ purchase decisions. Meat color stability is influenced by muscle source, and muscle-specific color stability has been investigated extensively in traditional livestock. In contrast, limited scientific information is available on this phenomenon in game meats. Fallow deer (Dama dama) is a game species having significant market potential. The objective of the present study was to examine the color stability of three economically important muscles in fallow deer carcasses, namely infraspinatus (IS), longissimus lumborum (LL), and biceps femoris (BF).Materials and MethodsThe IS, LL, and BF muscles were removed from both sides of six (n = 6) female fallow deer carcasses 24 h post-mortem. The muscles were vacuum-packaged and stored at 2°C. After 48 h, the muscles were fabricated into twelve 2.5-cm steaks. The steaks were placed in trays, aerobically over-wrapped, and stored at 2°C for 8 d. Meat pH, instrumental color (L*, a*, and b* values), color stability (R630/580; ratio of reflectance at 630 nm and at 580 nm), surface myoglobin redox forms, metmyoglobin reducing activity (MRA), and lipid oxidation were measured on 0, 1, 2, 4, 6, and 8 d. Data were analyzed using mixed model repeated measures ANOVA, with carcass as random effect, and muscle and time as fixed effects.ResultsThe IS steaks had the greatest (P < 0.05) pH, whereas the LL steaks exhibited the lowest (P < 0.05) pH throughout the storage. IS demonstrated greater (P < 0.05) L* values (lightness) than BF and LL during the storage. While a* values (redness) decreased (P < 0.05) in all muscles during storage, IS consistently exhibited greater (P < 0.05) a* values than LL and BF. In addition, during the storage, IS demonstrated greater (P < 0.05) b* values (yellowness), R630/580, MRA, and ferrous myoglobin forms (oxymyoglobin and deoxymyoglobin) than the LL and BF counterparts. Moreover, lipid oxidation and surface metmyoglobin content were lower (P < 0.05) in IS than in LL and BF.ConclusionThe findings of this study indicated that IS muscle of fallow deer is more color-stable than the LL and BF counterparts during refrigerated storage under aerobic conditions. The game industry may employ muscle-specific strategies to process and improve marketability of fresh meat from fallow deer.

Synthesis of Co(II)-NO(-) Complexes and Their Reactivity as a Source of Nitroxyl.

Metal-nitroxyl (M-HNO/M-NO(-)) coordination units are found in denitrification enzymes of the global nitrogen cycle, and free HNO exhibits pharmacological properties related to cardiovascular physiology that are distinct from nitric oxide (NO). To elucidate the properties that control the binding and release of coordinated nitroxyl or its anion at these biological metal sites, we synthesized {CoNO}(8) (1, 2) and {CoNO}(9) (3, 4) complexes that contain diimine-dipyrrolide supporting ligands. Experimental (NMR, IR, MS, EPR, XAS, XRD) and computational data (DFT) support an oxidation state assignment for 3 and 4 of high spin Co(II) (SCo = 3/2) coordinated to (3)NO(-) (SNO = 1) for Stot = 1/2. As suggested by DFT, upon protonation, a spin transition occurs to generate a putative low spin Co(II)-(1)HNO (SCo = Stot = 1/2); the Co-NO bond is ∼0.2 Å longer, more labile, and facilitates the release of HNO. This property was confirmed experimentally through the detection and quantification of N2O (∼70% yield), a byproduct of the established HNO self-reaction (2HNO → N2O + H2O). Additionally, 3 and 4 function as HNO donors in aqueous media at pH 7.4 and react with known HNO targets, such as a water-soluble Mn(III)-porphyrin ([Mn(III)(TPPS)](3-); TPPS = meso-tetrakis(4-sulfonatophenyl)porphyrinate) and ferric myoglobin (metMb) to quantitatively yield [Mn(TPPS)(NO)](4-) and MbNO, respectively.

Globins Scavenge Sulfur Trioxide Anion Radical

Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 10(6) m(-1) s(-1), respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 10(6) m(-1) s(-1), respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP(+)-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.

Tryptophan-to-heme electron transfer in ferrous myoglobins

It was recently demonstrated that in ferric myoglobins (Mb) the fluorescence quenching of the photoexcited tryptophan 14 (*Trp(14)) residue is in part due to an electron transfer to the heme porphyrin (porph), turning it to the ferrous state. However, the invariance of *Trp decay times in ferric and ferrous Mbs raises the question as to whether electron transfer may also be operative in the latter. Using UV pump/visible probe transient absorption, we show that this is indeed the case for deoxy-Mb. We observe that the reduction generates (with a yield of about 30%) a low-valence Fe-porphyrin π [Fe(II)(porph(●-))] -anion radical, which we observe for the first time to our knowledge under physiological conditions. We suggest that the pathway for the electron transfer proceeds via the leucine 69 (Leu(69)) and valine 68 (Val(68)) residues. The results on ferric Mbs and the present ones highlight the generality of Trp-porphyrin electron transfer in heme proteins.