Myoglobin Derivatives Research Articles

The mechanism of Leuconostoc mesenteroides subsp. IMAU:80679 in improving meat color: Myoglobin oxidation inhibition and myoglobin derivatives formation based on multi enzyme-like activities.

The Leuconostoc mesenteroides subsp. IMAU:80679 (LM) was chosen for its superior capability in enhancing redness, and was incubated in a broth system containing metmyoglobin (MetMb) to investigate its mechanisms for color improvement. The a* value of LM group reached its highest level of 52.75±1.04 at 24h, significantly higher than control of 19.75±0.6 (p<0.05). The addition of LM could inhibit myoglobin oxidation to some extent. Meanwhile, higher content of nitrosylmyoglobin (NOMb) and Zn-protoporphyrin (Znpp) were observed in LM samples during the whole incubation period. Furthermore, enzymatic activity and encoded genes related to MetMb reduction and pigment formation were determined to explain its possible mechanism on color enhancement. Finally, by extracting crude enzymes and adding them to meat batters, the redness of crude enzyme group was comparable to that achieved with 20ppm nitrite, providing a potential method on compensating for nitrite/nitrate substitution in meat products.

Potential application of lactic acid bacteria for controlling discoloration in tuna (Thunnus orientalis)

Pacific bluefin tuna (Thunnus orientalis) is a highly valued seafood consumed globally. The red color of tuna meat is considered a crucial indicator of its freshness and value, influencing consumer purchasing decisions. Therefore, the present study aimed to investigate the effect of lactic acid bacteria (LAB) isolated from traditional Korean fermented seafood (jeotgal) on preventing the discoloration of tuna meat during storage. Thirty-six strains of LAB (M1–M36) were isolated and examined for superior antioxidant and metmyoglobin conversion activities. Two strains, M1 (Lactococcus lactics) and M8 (Leuconostoc citreum), were selected. Subsequently, the tuna meat was treated with the cell-free supernatant (CFS) of the selected LAB strains and stored at 4 °C. Treatment with CFS maintained a significantly high level of redness (a*) in the tuna meat for up to 48 h; furthermore, this tendency was consistent with the relative quantification of myoglobin derivatives. The oxymyoglobin content in CFS-treated tuna meat remained above 60% for up to 60 h. Conversely, the metmyoglobin content in the untreated group reached over 60% only after 48 h, resulting in complete discoloration. Conclusively, this study demonstrated that LAB-CFS can prevent tuna meat discoloration during refrigerated storage, indicating its potential for maintaining the quality and color of tuna meat.

Effect of the exposure to oxidation and malondialdehyde on turkey and rabbit meat protein oxidative stability.

The present study aimed at evaluating the effect of the exposure to a strong oxidative environment (100mM NaClO) and the concurrent incubation with different malondialdehyde (MDA) concentrations (0 to 5mM) on protein carbonylation, free thiol groups, total heme pigments, and on the relative concentration of the different myoglobin (Mb) derivatives in turkey thigh and rabbit hind leg meat to elucidate their eventual role in inducing oxidative modifications on the protein fraction. With regard to turkey meat, the addition of a strong oxidant resulted in remarkably higher (P < 0.001) carbonyls along with a reduction in free thiol groups (which become undetectable). The relative concentration of MbO2 and MetMb was significantly affected (P < 0.001) and total heme pigment was reduced by 62% when fresh meat is compared to its oxidized counterpart. The addition of MDA 2.5mM (or greater) resulted in a 1.4-fold increase in carbonyls and a tendency (P = 0.07) has been observed for free thiol groups whose content decreased as the MDA level increased. Overall, the aforementioned results are in agreement with those obtained on rabbit hind leg meat in which a three-fold increase (P < 0.001) in carbonyls and a remarkably higher (P < 0.001) MetMb content coupled with a 53% reduction in free thiol groups were found. No significant differences ascribable to MDA addition were found. In conclusion, exposing turkey and rabbit meat to the same environment resulted in distinctive oxidative changes that might be related to the compositional traits and a species-specific susceptibility of the meat toward oxidation. PRACTICAL APPLICATION: Protein and lipid oxidation have been investigated in different meat types; nevertheless, the knowledge concerning the interaction between these two phenomena is limited. According to their compositional traits (high Polyunsaturated fatty acids (PUFA) and heme pigment content), turkey and rabbit leg meats are particularly susceptible to oxidation. Providing information concerning the oxidative stability of turkey thigh and rabbit hind leg meat exposed to strong oxidative conditions and to a concurrent increasing content of a secondary product of lipid oxidation, the findings of the present study can be useful when proper processing strategies and storage conditions have to be implemented for manufacturing processed products.

Diffuse reflectance spectroscopy to quantify the met-myoglobin proportion and meat oxygenation inside of pork and beef

The potential of diffuse reflectance spectroscopy (DRS) to quantify the met-myoglobin (met-Mb) proportion and meat oxygenation inside of pork and beef was examined. First, reflection spectra were obtained from pork (n = 52) and beef (n = 43) samples under fresh and stored conditions. Second, the DRS algorithm was applied to the reflectance spectra to calculate the met-Mb proportion and oxygenation of the meat. Lastly, a regression model was developed showing the change in the met-Mb proportion and oxygenation during met-Mb formation and degradation. A linear relationship existed between the DRS-based computed data and the known met-Mb proportion with a high correlation (R2 = 0.9999) and a low error (0.86%). Measurement of the meat samples revealed a linear increment of the met-Mb proportion (R2 = 0.77) and a quadratic change in the oxygenation (R2 = 0.44) during the met-Mb formation process. This study demonstrated the ability of DRS to quantitatively analyze the relative content of myoglobin derivatives in both pork and beef.

Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives

As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.

Conversion of Metmyoglobin to Bright Red Myoglobin Derivatives by Lactic Acid Bacteria

The aim of this research is to investigate the ability of lactic acid bacteria (LAB) isolated from Chinese dried sausage to convert metmyoglobin into bright red myoglobin derivatives in Mann-Rogosa-Sharp model systems. Of 70 LAB tested, one strain had a typical pink colour (judged by visual inspection) than the control samples and was identified as Weissella cibaria by serial biochemical tests and 16S rRNA analysis. This study provides a potential method for nitrite substitution by preserving or improving the color of meat products.

Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

Resonance Raman (rR) spectroscopy is generally useful to interrogate unstable intermediates that arise in the enzymatic cycles of heme enzymes, such as peroxo-, hydroperoxo- and ferryl species. While it is typically difficult to trap these elusive intermediates in solution, the cryoradiolysis approach offers a way to successfully overcome these obstacles. The present work employs modified derivatives of horse heart myoglobin to demonstrate the methodology and utility of a combination of rR and cryoradiolysis for detection and structural characterization of Fe―O―O, Fe―O―O―H and FeO fragments of such reactive species. First, the effect of active site mutations on the Fe―O―O fragments was investigated; specifically, the His64Leu, Val68Ser and Ile107Ala in the distal pocket and Ser92Ala, Ser92Leu and His97Phe on the proximal side. The current work has permitted documentation of the wavenumbers of the ν(Fe―O) stretching modes of the relatively unstable dioxygen adducts of these mutants, some of them previously unreported. Significantly, applying the cryoradiolysis approach has yielded previously unavailable vibrational spectroscopic data for the peroxo-, hydroperoxo and ferryl derivatives of these mutants. Of the six mutations investigated, only two, the H64L and V68S, replacements had a significant effect on the structure and stability of these unstable intermediates. In addition to these manipulations of the heme active site environment, the study of myoglobins bearing modified hemes was also undertaken; namely, myoglobins reconstituted with mesoheme and diformyl deuteroheme. The results show that the heme equatorial electronic effects significantly influence the stability and internal modes of the Fe―O―O and FeO fragments of these unstable oxy intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

Organometallic myoglobins: Formation of Fe–carbon bonds and distal pocket effects on aryl ligand conformations

Bioorganometallic Fe–C bonds are biologically relevant species that may result from the metabolism of natural or synthetic hydrazines. The molecular structures of four new sperm whale mutant myoglobin derivatives with Fe–aryl moieties, namely H64A–tolyl-m, H64A–chlorophenyl-p, H64Q–tolyl-m, and H64Q–chlorophenyl-p, have been determined at 1.7–1.9Å resolution. The structures reveal conformational preferences for the substituted aryls resulting from attachment of the aryl ligands to Fe at the site of net –NHNH2 release from the precursor hydrazines, and show distal pocket changes that readily accommodate these bulky ligands.

Formation of red myoglobin derivatives and inhibition of spoilage bacteria in raw meat batters by lactic acid bacteria and Staphylococcus xylosus

Abstract The conversion of metmyoglobin (MbFeIII) to red myoglobin derivatives by bacteria and their effect on the inhibition of spoilage organisms in raw meat batters as protective cultures were investigated. Culture-dependent and culture-independent (PCR-denaturing gradient gel electrophoresis) methods were employed to reveal the microbial populations. The results from visible absorption and electron spin resonance spectroscopy analysis showed that Staphylococcus xylosus and Lactobacillus fermentum could convert MbFeIII into nitrosylmyoglobin (MbFeIINO), while oxymyoglobin (MbFeIIO2) was formed by Lactobacillus curvatus and Lactobacillus plantarum in model systems. And the red colour was also observed in the meat batters inoculated with L. fermentum and S. xylosus. Staphylococcus spp., Carnobacterium sp., Lactobacillus sp., Brochothrix thermosphacta and Enterococcus faecalis were prevalent in control and nitrite-cured meat batters in tray packaging during refrigeration, whereas lactic acid bacteria and Staphylococcus were observed to be the predominant microbial flora in the samples inoculated with Lactobacillus sakei and S. xylosus, respectively. Most of the spoilage bacteria were inhibited by the two bacterial species. This study provides a potential method for nitrite substitution by improving meat colour and inhibiting spoilage bacteria in meat curing.

Resonance Raman determination of vinyl group disposition in different derivatives of native myoglobin and its heme‐disoriented form

In heme‐reconstituted heme proteins, the heme inserts such that some of the heme is rotated 180° about the α–γ meso axis, eventually equilibrating to native conformations. Also, proteins from different species may naturally possess both conformers. Resonance Raman (RR) spectroscopy is effective in signaling this conformational heterogeneity, detecting altered interactions with active site residues. While shifts of the vibrational modes of the two vinyl groups could be readily detected, they could not be assigned to a particular vinyl group. This deficiency is alleviated by employing a specifically labeled protoheme isotopomer, wherein only the 4‐vinyl group is labeled (i.e. –CH=C2H2), providing a spectral editing mechanism for selectively tracking the individual vinyl groups. The RR spectral data acquired here for the met‐, deoxy‐, and ferrous CO adduct of myoglobin, along with their ‘flipped heme’ isomers, provide convincing evidence for the anticipated effects of ‘swapping’ the environments of the two vinyl groups; i.e., in the native form, the 2‐vinyl group assumes a more out‐of‐plane orientation with respect to the pyrrole plane than does the (nearly in‐plane) 4‐vinyl group, whereas in the ‘reversed’ orientation, the 4‐vinyl group now assumes a position that is more out‐of‐plane than the newly positioned 2‐vinyl group. The ability of RR spectroscopy to document such differences in the orientation of the vinyl substituents is made important by the fact that changes in the disposition of these groups has long been known to carry functional consequences. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Equations for spectrophotometric determination of relative concentrations of myoglobin derivatives in aqueous tuna meat extracts

The percentage of metmyoglobin (%metMb) in aqueous meat extracts of bigeye and bluefin tuna and beef samples were estimated using previously reported equations derived from the absorption spectra of horse Mb. The results demonstrate that in an aqueous extract, the difference in %metMb estimated by the different equations was negligible for beef samples. Conversely, in an aqueous tuna extract, different %metMb values were obtained with the different equations. The discrepancy in the tuna sample results might be due to differences in absorption spectra for horse and tuna Mb. Therefore, a new set of equations derived from the absorption spectra of bigeye tuna Mb, reported by Matsuura and Hashimoto (1955), was established. The accuracy of the proposed equations was compared with the cyanmetmyoglobin (cyanmetMb) method. The results show that the total Mb concentrations estimated by our proposed equations were in good agreement with the results obtained by the conventional cyanmetMb method (R2=0.984). Therefore, the new set of proposed equations is valid for the spectrophotometric determination of the relative proportions of Mb derivatives and total Mb concentration in aqueous tuna meat extracts.

Polarized X-ray Absorption Near-Edge Structure Spectroscopy of Neuroglobin and Myoglobin Single Crystals

Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-neuroglobin (NgbCO) single crystals have been collected and compared with a number of derivatives of sperm whale myoglobin (Mb), that is, the nitrosyl (MbNO) and deoxy (Mb) derivatives, the previously reported cyanomet (MbCN) and carbonmonoxy (MbCO) derivatives, and the cryogenic photoproduct of MbCO (Mb·CO). The single crystals under study exhibit a strong XANES angular dichroism which allows the heme geometry of each sample to be analyzed with extremely high accuracy via the full multiple scattering (MS) approach. The results of two alternative methods to undergo the MS analysis have been compared with high resolution X-ray diffraction (XRD) data and with X-ray absorption spectroscopy (XAS) data in solution. As a result of the present analysis, the Fe-heme structure in solution and in the cryo-trapped NgbCO single crystal (which cracks at room temperature) are the same. Accordingly, the residual energy involved in the protein relaxation responsible of crystal cracking at room temperature after CO binding does not reside in the heme pocket. A combined approach (polarized XANES and XRD) is suggested to be applied on the same single crystals of metalloproteins at opportunely equipped synchrotron beamlines.

Resonance Raman spectroscopic studies of hydroperoxo derivatives of cobalt-substituted myoglobin

Recent progress in generating and stabilizing reactive heme protein enzymatic intermediates by cryoradiolytic reduction has prompted application of a range of spectroscopic approaches to effectively interrogate these species. The impressive potential of resonance Raman spectroscopy for characterizing such samples has been recently demonstrated in a number of studies of peroxo- and hydroperoxo-intermediates. While it is anticipated that this approach can be productively applied to the wide range of heme proteins whose reaction cycles naturally involve these peroxo- and hydroperoxo-intermediates, one limitation that sometimes arises is the lack of enhancement of the key intraligand ν(O–O) stretching mode in the native systems. The present work was undertaken to explore the utility of cobalt substitution to enhance both the ν(Co–O) and ν(O–O) modes of the CoOOH fragments of hydroperoxo forms of heme proteins bearing a trans–axial histidine linkage. Thus, having recently completed RR studies of hydroperoxo myoglobin, attention is now turned to its cobalt-substituted analogue. Spectra are acquired for samples prepared with 16O 2 and 18O 2 to reveal the ν(M–O) and ν(O–O) modes, the latter indeed being observed only for the cobalt-substituted proteins. In addition, spectra of samples prepared in deuterated solvents were also acquired, providing definitive evidence for the presence of the hydroperoxo-species.

A multispectral imaging technique to determine concentration profiles of myoglobin derivatives during meat oxygenation

We have designed a multispectral imaging acquisition system to measure the relative concentration values of myoglobin forms inside meat during oxygenation. Images at 474, 525, 572 and 610 nm are used to compute the concentration of reduced myoglobin, oxymyoglobin and metmyoglobin with a spatial resolution of 0.0125 mm/pixel. From these images, pigment concentration profiles as a function of oxygenation time and depth beneath the surface were obtained. A model describing the diffusion of oxygen and the consumption of reduced myoglobin is numerically tested versus the measured concentration profiles. The model accurately fits the data with a Mean Root Squared Error equal to 0.253%. Precise definitions for position and width of pigment layers based in concentration profiles are given. The results suggest that multispectral imaging techniques combined with precise control and measurement of sample temperature and oxygen partial pressure will permit detailed studies of the myoglobin chemistry during oxygenation.

Asymmetric band profile of the Soret band of deoxymyoglobin is caused by electronic and vibronic perturbations of the heme group rather than by a doming deformation

We measured the Soret band of deoxymyoglobin (deoxyMb), myoglobin cyanide (MbCN), and aquo-metmyoglobin (all from horse heart) with absorption and circular dichroism (CD) spectroscopies. A clear non-coincidence was observed between the absorption and CD profiles of deoxyMb and MbCN, with the CD profiles red- and blueshifted with respect to the absorption band position, respectively. On the contrary, the CD and absorption profiles of aquametMb were nearly identical. The observed noncoincidence indicates a splitting of the excited B state due to heme-protein interactions. CD and absorption profiles of deoxyMb and MbCN were self-consistently analyzed by employing a perturbation approach for weak vibronic coupling as well as the relative intensities and depolarization ratios of seven bands in the respective resonance Raman spectra measured with B-band excitation. The respective B(y) component was found to dominate the observed Cotton effect of both myoglobin derivatives. The different signs of the noncoincidences between CD and absorption bands observed for deoxyMb and MbCN are due to different signs of the respective matrix elements of A(1g) electronic interstate coupling, which reflects an imbalance of Gouterman's 50:50 states. The splitting of the B band reflects contributions from electronic and vibronic perturbations of B(1g) symmetry. The results of our analysis suggest that the broad and asymmetric absorption band of deoxyMb results from this band splitting rather than from its dependence on heme doming. Thus, we are able to explain recent findings that the temperature dependences of CO rebinding to myoglobin and the Soret band profile are uncorrelated[Ormos et al., Proc. Natl. Acad. Sci U.S.A. 95, 6762 (1998)].

Sugar-derived Glasses Support Thermal and Photo-initiated Electron Transfer Processes over Macroscopic Distances

Trehalose-derived glasses are shown to support long range electron transfer reactions between spatially well separated donors and protein acceptors. The results indicate that these matrices can be used not only to greatly stabilize protein structures but also to facilitate both thermal and photo-initiated hemeprotein reduction over large macroscopic distances. To date the promise of exciting new protein-based technologies that can harness the exceptional tunability of protein functionality has been significantly thwarted by both intrinsic instability and stringent solvent/environment requirements for the expression of functional properties. The presented results raise the prospect of overcoming these limitations with respect to incorporating redox active proteins into solid state devices such as tunable batteries, switches, and solar cells. The findings also have implications for formulations intended to enhance long term storage of biomaterials, new protein-based synthetic strategies, and biophysical studies of functional intermediates trapped under nonequilibrium conditions. In addition, the study shows that certain sugars such as glucose or tagatose, when added to redox-inactive glassy matrices, can be used as a source of thermal electrons that can be harvested by suitable redox active proteins, raising the prospect of using common sugars as an electron source in solid state thermal fuel cells.

Low frequency resonance Raman spectra of isolated α and β subunits of hemoglobin and their deuterated analogues

In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups.

Bright Red Color Formation of Cooked Pork Loin Cured with Lactic Acid Bacteria Starter Culture without Adding Nitrite During Low-Temperature Storage

The effects of lactic acid bacteria used as a substitute for nitrite for meat processing were studied. We added lactic acid bacteria starter culture to brine to cure pork loin without nitrite. The cured pork loin was then smoked, sliced, vacuum-packaged, and stored at 2°C for two weeks and at 7°C for additional weeks. A preservative effect of the added lactic bacteria was observed. A homo-fermentative lactic acid bacteria Lactobacillus sakei M32 was a good starter culture to control the microflora of products without nitrite. Bright red color development and propagation between some samples were observed during the cold storage in vacuum packages. This color development is not yet practical for industrial application as a substitute for nitrite. The samples, which had turned clear red in cold storage after two weeks, were further analyzed. In these samples, free arginine markedly decreased, while other free amino acids slightly increased. Although the residual nitrite in the samples was less than one ppm, the degree of color formation of the samples was similar to that of the positive nitrite-added control samples. The results suggested formation of bright red myoglobin derivatives, such as nitrosylmyoglobin from arginine, in the samples.

Characterisation of myoglobin from sardine ( Sardinella gibbosa) dark muscle

Myoglobin from the dark muscle of sardine ( Sardinella gibbosa) with the molecular weight of 15.3 kDa was isolated and characterised. The different myoglobin derivatives exhibited varying thermal unfolding characteristics. Deoxymyoglobin showed a single distinct endothermic peak at 74.5 °C, whereas two transition temperatures were noticeable for oxymyoglobin (64.5 and 78.4 °C) and metmyoglobin (59.0 and 76.0 °C). The spectrum of deoxymyoglobin and oxymyoglobin had absorption bands at 739, 630, 575, 500 and 405 nm, while the disappearance of the peak at 575 nm was found in the spectrum of metmyoglobin. The soret peak of all derivatives was noticeable at 405 nm. The autoxidation of myoglobin became greater at very acidic or alkaline conditions as evidenced by the formation of metmyoglobin, the changes in tryptophan fluorescence intensity as well as the disappearance of soret absorption. The higher temperature, particularly above 40 °C, and the longer incubation time induced the higher metmyoglobin formation as well as the conformational changes.

Effects of systematic peripheral group deuteration on the low-frequency resonance Raman spectra of myoglobin derivatives.

Resonance Raman spectra are reported for a series of systematically deuterated analogues of myoglobin in its deoxy state as well as for its CO and O(2) adducts. Specifically, the myoglobin samples studied are those that have been reconstituted with deuterated protoheme analogues. These include the methine deuterated, protoheme-d4; analogue bearing C(2)H(3) groups at the 1, 3, 5, and 8 positions, protoheme-d12; the species bearing C(2)H(3) groups at the 1 and 3 positions only, 1,3-protoheme-d6; and the species bearing C(2)H(3) groups at the 5 and 8 positions only, 5,8-protoheme-d6. While the results are generally consistent with previously reported data for synthetic metalloporphyrin models and previous studies of labeled heme proteins, the high-quality low-frequency RR data reported here reveal several important aspects of these low-frequency modes. Of special interest is the fact that, using the two d6-protoheme analogues, it is shown that certain modes are apparently localized on particular pyrrole rings, while others are localized on different rings; i.e., several of these low-frequency modes are localized on one side of the heme.