DOCSLIB.ORG

Quantification of NADH-Cytochrome B5 Reductase (Metmyoglobin-Reducing Enzyme) in Bovine Skeletal Muscle by an Immunoblotting Assay

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Quantification of NADH-Cytochrome B5 Reductase (Metmyoglobin-Reducing Enzyme) in Bovine Skeletal Muscle by an Immunoblotting Assay Quantification of NADH-Cytochrome b5 Reductase (Metmyoglobin-Reducing Enzyme) in Bovine Skeletal Muscle by an Immunoblotting Assay Keizo ARIHARA, Makoto ITOH and Yo KONDO School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi 034 (Received May 8, 1996) Abstract Quantitative determination method of NADH-cytochrome b5 reductase (me- tmyoglobin-reducing enzyme) in bovine skeletal muscle was developed by combining SDS-PAGE and immunoblotting techniques. Recoveries of the enzyme by this assay ranged from 90.0 to 94.4%. The content of NADH-cytochrome b5 reductase in bovine skeletal muscle was estimated to be 13.8±2.6(9.0-18.2)μg/g tissue (n=19). Also, a quantitative assay for determining cyto- chrome b5 (an electron transfer mediator for metmyoglobin reduction) was similarly established and cytochrome b5 content in bovine skeletal muscle was estimated to be 59.0±20.9μg/g tissue. The quantitative data of NADH-cytochrome b5 reductase in this study supported our previous hypothesis that the enzyme plays a critical role in preventing metmyoglobin accumulation in muscles. Anim. Sci. Technol. (Jpn.) 68 (1): 29-33, 1997 Key words: NADH-cytochrome b5 reductase, Metmyoglobin-reducing enzyme, Bovine skeletal muscle, Immunoblotting, Quantitative assay Myoglobin is responsible for oxygen storage muscles, we have demonstrated the signifi- and transport in living muscles14). However, it cance of NADH-cytochrome b5 reductase is easily oxidized to metmyoglobin, resulting in (NADH: cytochrome b5 oxidoreductase, EC 1.6. the impairment of physiological functions of 2.2.) for preventing metmyoglobin accumula- myoglobin. Also, myoglobin plays an impor- tion. Until recently, this enzyme in muscle tant role in post mortem change of meat was also called metmyoglobin reductase2,3,11). color2,8,13), an important sensory property We have proposed a new pathway that NADH affecting consumer acceptability, because meat -cytochrome b5 reductase reduces metmyo - color is dependent on the amount and the globin to myoglobin via either OM cytochrome chemical state of myoglobin. Desirable b or cytochrome b5 as an electron transfer cherry red color of fresh meat turns brown due mediator2,5,6). This pathway would contribute to the accumulation of metmyoglobin during to keep the chemical state of the heme of myo- storage. Because of these reasons, preventing globin in ferrous state in maintaining normal metmyoglobin formation is critical in keeping physiological functions of myoglobin in normal physiological functions of muscle and muscles. Therefore, this pathway would play in keeping fresh meat color. a very important role. In a series of our studies2-7) concerning Thus, it is desirable to establish a reliable metmyoglobin-reducing enzyme systems in method to determine NADH-cytochrome b5 Anim. Sci. Technol. (Jpn.) 68 (1): 29-33 29 1997 ARIHARA, ITOH and KONDO reductase content in order to make a thorough b5, reductase and cytochrome b5 previously investigation in proving the pathway. This raised in rabbits6) were used in this study, enzyme is present as a tightly membrane- Antibodies in the serum were purified by bound form in most tissues (e.g. liver, brain, affinity chromatography on CNBr-Sepharose 4 placenta, lung, muscle)15,16), and its activity is B coupled to the purified bovine NADH-cyto- relatively unstable and inactivated during ex- chrome b5 reductase or cytochrome b5 accord- traction procedures3). Since it seems likely ing to the manufacturer's protocols (Pharmacia that the concentration of endogenous interme- Fine Chemicals, Uppsala, Sweden). The diate acceptor (cytochrome b5 or OM cyto- specifity of antibodies was confirmed by an chrome b) is not saturated in those tissues, immunoblotting technique. differences in its concentration could be a SDS-PAGE and Immunoblotting: cause of the differences in the activity of the Determination of NADH-cytochrome b5 enzyme at equal enzyme concentrations. reductase and cytochrome b5 was carried out as Also, physiological intermediate acceptors for follows. SDS-PAGE was performed according the assay of enzyme activity are not available to the method of Laemmli10), using 12.5% ac- commercially. Because of these reasons, it has rylamide separating gel. Muscle samples (0.1 been difficult to examine the exact activity and g) were minced with scissors and dispersed in quantity of the enzyme, in muscles. In this Laemmli's sample buffer (1.9ml) with Potter's study, we determined the content of NADH- homogenizer for 1min before heating (100℃, 3 cytochrome b5 reductase and cytochrome b5 in min). The homogenate was centrifuged at bovine skeletal muscle by immunoblotting 5,000×g for 5min and its supernatant (10μl) techniques. was used for SDS-PAGE. Following the fractionation by SDS-PAGE, proteins were Materials and Methods transferred from the gel to PVDF membranes Bovine skeletal muscle: (Clearblot P-membrane, Atto Co., Tokyo), as Bovine thigh muscle (biceps femoris) used for outlined by Kyhse-Andersen9), using a home establishing a quantitative method was ob- made horizontal electroblotting apparatus. tained immediately after slaughter with the Blotting was carried out with 25mM Tris-192 assistance of the laboratory of veterinary pa- mM glycine-20%(v/v) methanol buffer (pH8.3) thology staff, Kitasato University. The at 100mA for 60min. After electroblotting , muscle was stored at -20℃ until use. For the blots on the membrane were immunostained determination of NADH-cytochrome b5 using the primary antibody (anti-NADH-cyto- reductase content in beef, fresh thigh muscle chrome b5 reductase or anti-cytochrome b5, ap- samples were purchased from a local super- proximately 1μg IgG/ml) described above and market. an Bio-Rad Immun-Blot Assay Kit (Bio-Rad NADH-cytochrome b5 reductase and cyto- Laboratories, Richmond, CA, USA; secondary chrome b5: antibody, horseradish peroxidase-labelled goat NADH-cytochrome b5 reductase and cyto- anti-rabbit IgG; development reagent , 4- chrome b5 for immunization described below chloro-1-naphthol). All immunostainning and standard solutions for the assay were pre- procedures were performed according to the pared from bovine erythrocytes according to manufacturer's protocols . Pre-stained SDS- the procedures described in previous PAGE standards and SDS-PAGE low-molecu- papers3,4,6). lar-weight standards (Bio-Rad) were used to Antibodies: monitor the efficiency of electrophoretic trans - Antisera against bovine NADH-cytochrome fer of the enzyme and to determine the molecu - 30 Muscle Metmyoglobin-Reducing Enzyme lar weight of the enzyme, respectively. tration. From the standard curve shown in Absorbance and area of immunostained bands Fig. 1, the assay for this enzyme was quantita- on the membrane was measured by reflection tive over the entire range examined (4-128ng). mode at 570nm with a Shimadzu (Kyoto, As shown in Table 1, the recoveries of the com- Japan) chromato-scanner CS-930. ponent added to bovine skeletal muscle homo- genate demonstrated that the quantification Results and Discussion procedure of the present study is highly relia- After SDS-PAGE and immunoblotting, ble and gives satisfactory values. The deter- NADH-cytochrome b5 reductase in bovine skel- mination method for cytochrome b5 was also etal muscle was well recognized as a single established similarly with that for NADH-cy- band on the immunostained membrane in ac- tochrome b5 reductase. These results strongly cordance with the results obtained in the previ- indicate that the present quantitative method ous paper6). Thus, the immunological detec- based on the immunoblotting procedure is ap- tion method of the present study was proved to plicable for the determination of NADH-cyto- be highly specific for the enzyme. The molec- ular weight value (35,000) of NADH-cyto- Table 1. Recoveries of NADH-cytochrome b5 chrome b5 reductase, based on the position of reductase added to bovine skeletal muscle the immunostained band, was identical to that homogenates of a previous paper6). The optimum heating period and SDS concentration for the prepara- tion of SDS-PAGE samples were 3min and 2%, respectively. Quantification of the amount of the enzyme present in muscles was made by preparing a 1Purified enzyme perparation was added to 10μl of standard calibration curve by plotting the ab- sorbance of the purified NADH-cytochrome b5 muscle homogenate sample for SDS-PAGE. 2Average of three replicates . reductase preparation versus protein concen- Fig. 1. Calibration curve for standard NADH-cytochrome b5 reductase solution. A, calibration curve drawn from the peak area of densitogram (B); B, densitogram of the membrane immunostained by anti-NADH-cytochrome b5 reductase antibodies. 31 ARIHARA, ITOH and KONDO Table 2. Contents of NADH-cytochrome b5 reductase and cytochome b5 in bovine skeletal muscle determined by the immunoblotting procedure 1 Average of three replicates of each sample was used. chrome b5 reductase and cytochrome b5 content Acknowledgements in muscle. Averages of the contents of NADH-cyto- This work was supported in part by a Grant chrome b5 reductase and cytochrome b5 (elec- -in-Aid for Scientific Research (No. 02760165) tron-transfer mediator for metmyoglobin re- from the Ministry of Education, Science and duction)4) in bovine skeletal muscle (thighs) Culture of Japan. were estimated to be 13.5 and 59.0μg/g muscle, References respectively, by the present immunoblotting procedure (Table 2). 1) Antonini E, Brunori M. The derivatives of fer- In erythrocytes, it has been recognized that rous hemoglobin
Load more

Recommended publications
  • Strategies for Increasing Oxidative Stability of (Fresh) Meat Color
    O X I D A T I V E P R O C E S S E S I N M E A T Strategies for Increasing Oxidative Stability of (Fresh) Meat Color CAMERON FAUSTMAN*, W.K.M. CHAN, M.P. LYNCH and S.T. JOO Introduction moglobin and myoglobin) and for electron transport during respiration (cytochromes) in the living animal. Skeletal Fresh meat color is determined by the oxidation status muscles which obtain energy primarily by aerobic means of myoglobin. Several reviews of myoglobin chemistry and contain relatively high concentrations of heme proteins when meat color stability, including cured and/or cooked meat compared to those which depend primarily on anaerobic color, have been published (Livingston and Brown, 1981; glycolysis. Giddings, 1977; Faustman and Cassens, 1990a; Renerre, Of the heme proteins found in post-mortem skeletal 1990; Cornforth, 1994). The purpose of this presentation is muscle, myoglobin is the most important for considerations to emphasize recent research findings which impact the oxi- of meat color. The majority of hemoglobin found in the liv- dative stability of myoglobin in fresh meat. Specific atten- ing animal is lost during slaughter as a result of exsan- tion is given to metmyoglobin reduction and antioxidant ap- guination. Some blood is retained in meat (Fleming et al., proaches for minimizing oxymyoglobin oxidation. 1960) and Warriss and Rhodes (1977) estimated the aver- Maintenance of oxymyoglobin and thus a desirable age residual blood content of butcher’s meat to be 0.3%. appearance in fresh meat has significant economic impact. Han et al. (1994) recently published a procedure for deter- Liu et al.
    [Show full text]
  • Relationship Between Beef Colour and Myoglobin
    Relationship between beef colour and myoglobin Author: S.H.J.M . Dobbelstein Registration number: 790608185120 Subject code: P052-754 (24 stp) P052-274 (1 stp) Supervisors: Dr. Ir. E.U. Thoden van Velzen (A&F) Dr. Ir.J.P.H . Linssen (WUR) Examiner: Prof. Dr. Ir. M.A.J.S. van Boekel Report 474 Preface This thesis is part of my Master in Food Technology at Wageningen UR. This subject was carried out at the Quality in Chains department of Agrotechnology & Food Innovations (A&F) in co­ opération with the Product Design and Quality Management Group of Wageningen UR. I worked on this thesis from January till August 2005 at A&F. During my study I became interested in Meat Science and the subject of this thesis fits perfecdy to my interests. During my stay at A&F I had all freedom and responsibility to conduct my research. This made I really enjoyed working on this thesis. Moreover, it increased my knowledge of Meat Science very much. I would like to thank Ulphard Thoden van Velzen, my supervisor at A&F, and Jozef Linssen, my supervisor at the University, for their help during this study. I also want to thank Ronald Holtmaat from ProMessa (Deventer, NL) who kindly provided the meat for this study. Furthermore I would like to thank Aart Zegveld, Dianne Somhorst and Janny Slotboom who helped me with my practical work at A&F. ©Agrotechnology &Foo d Innovations B.V .Membe r of Wageningen UR Abstract The most important quality attribute of fresh beef is its colour.
    [Show full text]
  • Myoglobin from Equine Skeletal Muscle
    Myoglobin from equine skeletal muscle Catalog Number M0630 Storage Temperature –20 C CAS RN 100684-32-0 Precautions and Disclaimer This product is for R&D use only, not for drug, Product Description household, or other uses. Please consult the Safety Molecular mass:1 17.6 kDa Data Sheet for information regarding hazards and safe Extinction coefficient:2 EmM = 12.92 (555 nm) handling practices. pI:3 7.3 (major component) and 6.8 (minor component) Preparation Instructions Myoglobin from horse skeletal muscle is a single chain This protein is soluble in water (10 mg/ml), yielding a heme protein containing 153 amino acid residues. It clear, red brown solution. posesses no disulfide bridges or free -SH groups. Myoglobin contains 8 variously sized right-handed References helical regions, joined by non-ordered or random coil 1. Darbre, P.D. et al., Comparison of the myoglobin of regions. These 8 helices (A, B, C, D, E, F, G, and H) the zebra (Equus burchelli) with that of the horse are folded back on top of one another, and the heme is (Equus cabalus). Biochim. Biophys. Acta, 393(1), situated between helices E and F. The heme is almost 201-204 (1975). totally buried. Only the edge carrying the two 2. Bowen, W.J., The absorption spectra and extinction hydrophylic propionic acid groups is exposed. The coefficients of myoglobin. J. Biol. Chem., 179, 235- heme is held in position by a coordinating complex 245 (1949). between the central Fe(II) atom and 2 histidine residues 3. Radola, B.J., Isoelectric focusing in layers of (on helices E and F, respectively).
    [Show full text]
  • 1 Hemoglobin Catalyzes ATP-Synthesis in Human
    Hemoglobin catalyzes ATP-synthesis in human erythrocytes: A murburn model Abhinav Parashar*, Vivian David Jacob, Daniel Andrew Gideon, Kelath Murali Manoj* *Corresponding authors, Satyamjayatu: The Science & Ethics Foundation, Snehatheeram, Kulappully, Shoranur-2, Kerala, India-679122. [email protected] Abstract: Blood hemoglobin (Hb) is the most abundant globular protein in humans, known to transport oxygen. Erythrocytes have ~10-3 M concentration levels of ATP in steady-state and we estimate that this high cannot be formed from 10-4 - 10-7 M levels of precursors via substrate- level phosphorylation of glycolysis. To account for this discrepancy, we propose that Hb serves as a ‘murzyme’ (a redox enzyme working along the principles of murburn concept), catalyzing the synthesis of the major amounts of ATP found in erythrocytes. This proposal is along the lines of our earlier works demonstrating DROS (diffusible reactive oxygen species) mediated ATP- synthesis as a thermodynamically and kinetically viable mechanism for physiological oxidative phosphorylation. We support the new hypothesis for Hb with theoretical arguments, experimental findings of reputed peers and in silico explorations. Using in silico methods, we demonstrate that adenoside nucleotide and 2,3-bisphosphoglycerate (2,3-BPG) binding sites are located suitably on the monomer/tetramer, thereby availing facile access to the superoxide emanating from the heme center. Our proposal explains earlier reported in situ experimental findings/suggestions of 2,3-BPG and ADP binding at the same locus on Hb. The binding energy is in the order of 2,3-BPG > NADH > ATP > ADP > AMP and agrees with earlier reports, potentially explaining the bioenergetic physiology of erythrocytes.
    [Show full text]
  • As Well As Whale Metmyoglobin in 0.1 M Deuterated Phosphate at Pd 7 and at 31'C
    NUCLEAR MAGNETIC RESONANCE STUDIES OF HEMOGLOBINS, III. EVIDENCE 1OR THE NONEQUIVALENCE OF a- AND (3-CHAINS IN AZIDE DERIVATIVES OF AME'TIIEMOGLOBINS* BY DONALD G. DAVIS, SAMUEL CHARACIHEt AND) CHIEN Ho DEPARTMENT OF BIOPHYSICS AND MICRtOBIOLOGY, UNJVI;RSI'IY' OlF Pl-ITT'SlBURGH, PITTSBURGH, PENNSYLVANIA Communicated by George Scatchard, June 12, 1969 Abstract.-Nuclear magnetic resonance spectroscopy (100-MN1Hz proton) was used to study the low-spin (S = 1/2) azide derivatives of human adult (a2(32), human fetal (C2Y2), Zurich (a2,f263 HiS - Arg), and horse (a2'(32') methemoglobins, as well as whale metmyoglobin in 0.1 M deuterated phosphate at pD 7 and at 31'C. The experimental results indicate that the azide-bound heme groups of the a- and (-chains in human adult methemoglobin and of the a- and 'y-chains in fetal methemoglobin are not equivalent. The affinity of the (3- or e-chain for azide ion appears larger than that of the a-chain. The nuclar magnetic resonance spectrum of hemoglobin Zfirich shows that the environment of the azide-heme complex in the abnormal (-chain is altered by the substitution of arginine for histidine in the (3-63 position, while the a-heme environment remains unaffected. Introduction.-The chief physiologic function of hemoglobin is to transport molecular oxygen from lung to tissues by virtue of its ability to combine revers- ibly with oxygen. Hemoglobin is a protein molecule consisting of four subunits (normally two a-chains and two (3-chains). Each of the four subunits has a heme group, and these heme groups are the "active centers" of hemoglobin.
    [Show full text]
  • AMSA Meat Color Measurement Guidelines
    AMSA Meat Color Measurement Guidelines Revised December 2012 American Meat Science Association http://www.meatscience.org AMSA Meat Color Measurement Guidelines Revised December 2012 American Meat Science Association 201 West Springfield Avenue, Suite 1202 Champaign, Illinois USA 61820 800-517-2672 [email protected] http://www.meatscience.org CONTENTS Technical Writing Committee .................................................................................................................... v Preface ..............................................................................................................................................................vi Section I: Introduction ................................................................................................................................. 1 Section II: Myoglobin Chemistry ............................................................................................................... 3 A. Fundamental Myoglobin Chemistry ................................................................................................................ 3 B. Dynamics of Myoglobin Redox Form Interconversions ........................................................................... 3 C. Visual, Practical Meat Color Versus Actual Pigment Chemistry ........................................................... 5 D. Factors Affecting Meat Color ............................................................................................................................... 6 E. Muscle
    [Show full text]
  • Processing Effects on Fresh and Frozen Meat Color Dale L
    Processing Effects on Fresh and Frozen Meat Color Dale L. Huffman* Introduction pletely filled with hydrophobic groups. Deep in the hy- drophobic interior is a cleft where heme is located. According In recent years there has been a considerable amount of to Adam (1976) the globin moiety does not assume its native interest in further processing of fresh meat. The primary con- conformation unless it is complexed with heme. The heme sideration in evaluating freshness is color. The purpose of this group serves to stabilize the configuration of the myoglobin manuscript is to assess the present status of processed fresh molecule. The heme is oriented in such a way that the vinyl meat color. groups are buried in the hydrophobic interior and the car- boxyl group of the propionic acids extend from the interior Myoglobin and color in fresh meat where they form part of the polar surface. Both the propionic Although a number of pigments are present in muscle, acid side chains are believed to be hydrogen bonded to myoglobin is generally the only pigment present in large serine, histidine, and arginine amino acid residues on the enough quantities to color meat. Hemoglobin constitutes globin moiety (Antonini and Brunori, 1971). According to 12-30% of the total muscle pigment. Since the two pigments Govindarajan (1973) the entire inner portion of the heme is have similar spectral properties, myoglobin alone is used as surrounded by side chains of nonpolar amino acids. It is due an index of fresh meat color. to these nonpolar surroundings that ferrous iron (Fef2) in The myoglobin content of muscle tissue varies not only be- myoglobin is able to reversibly combine with oxygen (Govin- tween species but between age group as well as muscle types darajan, 1973).
    [Show full text]
  • THE CHEMISTRY of HÆOGLOBIN and MYOGLOBIN in RELATION to the COLOR of MEAT DISSERTATION Presented in Partial Fulfillment Of
    THE CHEMISTRY OF HÆOGLOBIN AND MYOGLOBIN IN RELATION TO THE COLOR OF MEAT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By HOWARD NED DRAUDT, B.Sc., M.Sc. The Ohio State University 1955 Approved by: Adviser The Department of Agricultural Biochemistry TABLE OF CONTENTS Page INTRODUCTION ........................................... 1 LITERATURE SURVEY ....................................... 3 Myoglobin and Hemoglobin.... .................... 3 Methemoglobin or Metmyoglobin Formation ......... 7 The Action of Nitrites on Hemoglobin and Myoglobin ... 11 The Effect of pH on Curing ..... 18 The Effect of Reducing Agents in Meat Curing ....... 19 Heating and Hemochrome Formation... ................ 21 Color Loss in Cured Products ..... 2k EXPERIMENTAL PROCEDURE .................................. 27 Obj ect of the Investigation ......... 27 The Effect of Heating on Color Fixation and Color Stability.......... 29 Isolation of Metmj'-oglobin ........................ 37 Qualitative Experiments on the Effect of Possible Meat Components on Discoloration ...................... 39 Spectroscopy of the Pigment ...... 54 Manometric Experiments ....... 6l Preparation of Samples ........ 62 Warburg Experiments with Heat Fractionated Pigment ... 65 Gas Uptake in the Presence of Sodium Pyruvate ...... 70 Results of Warburg Experiments with Purified Pigment in which Nitrate and Nitrite were not Determined ..... 72 ii Page The Effect of Oleic Acid on Oxygen
    [Show full text]
  • Direct Observation of Ligand Migration Within Human Hemoglobin at Work
    Direct observation of ligand migration within human hemoglobin at work Naoya Shibayamaa,1, Ayana Sato-Tomitaa, Mio Ohkib, Kouhei Ichiyanagia, and Sam-Yong Parkc aDivision of Biophysics, Department of Physiology, Jichi Medical University, Tochigi, 329-0498 Shimotsuke, Japan; bResearch Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Didcot, Oxon OX11 0FA, United Kingdom; and cDrug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, 230-0045 Yokohama, Japan Edited by William A. Eaton, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, and approved January 24, 2020 (received for review August 8, 2019) Hemoglobin is one of the best-characterized proteins with respect His(E7) gate pathway (9, 10). Therefore, controversy remains as to structure and function, but the internal ligand diffusion pathways to whether the Xe-binding cavities play a functional role in Hb. remain obscure and controversial. Here we captured the CO For directly observing the ligand migration in Hb, X-ray migration processes in the tense (T), relaxed (R), and second relaxed crystallographic analysis of COHb after photolysis is obviously (R2) quaternary structures of human hemoglobin by crystallography preferred but is quite challenging and complex, due to the rel- using a high-repetition pulsed laser technique at cryogenic temper- atively low apparent quantum yield for the photolysis of COHb atures. We found that in each quaternary structure, the photo- (11), as well as the need to address the quaternary structure dissociated CO molecules migrate along distinct pathways in the α dependence of gas diffusion as predicted by MD simulations (7). and β subunits by hopping between the internal cavities with cor- In addition, excitation light transfer to the sample, together with related side chain motions of large nonpolar residues, such as the rapid kinetics of CO recombination, place limits on crystal α α β β 14Trp(A12), 105Leu(G12), 15Trp(A12), and 71Phe(E15).
    [Show full text]
  • Effect of Arginine-Hemoglobin on Color Stability of Ground Lamb During Storage
    EFFECT OF ARGININE-HEMOGLOBIN ON COLOR STABILITY OF GROUND LAMB DURING STORAGE Song Xuan1,2, Hou Chengli1, Wang Wenting1,2, Li Zheng1, Xin Li1, Wu Liguo1, Zhu Jie2, Zhang Dequan1, * (1 Institute of Food Science and Technology, Chinese Academy of Agricultural Science, Beijing 100193; 2 Laboratory of Biomechanics and Engineering, Institute of Biophysics and College of Science, Northwest A&F University, Yangling, Shaanxi 712100) *Corresponding author email: [email protected] Abstract – This study investigated the effect of arginine-hemoglobin on color stability of ground lamb during storage. Meat color, percentage of metmyoglobin (MetMb%) and thiobarbituric acid reactive substance (TBARS) were analyzed under different concentration (0, 0.15, 0.25, 0.5 g/kg) of arginine-hemoglobin during 7 days storage at 4±0.2 °C. The data showed that the arginine-hemoglobin treatments had lower TBARS and MetMb% compared with control. The redness (a* value) of treatments were higher than that in control during storage. In addition, the redness value increased as the arginine-hemoglobin concentration increased. The results suggested that the addition of arginine-hemoglobin could be an option in inhibiting the oxidation of myoglobin and fat in meat. Key Words-arginine, hemoglobin, color, ground lamb I. INTRODUCTION Meat color is one of the most important indexes to evaluate the acceptability of meat product. The color of meat and meat products are affected by the oxidation of myoglobin (Mb) and lipid. In addition, the nutrition and sensory of meat and meat products are also affected by oxidation of Mb and lipid [1]. Various methods have been attempted to inhibit or reduce the oxidation reaction in meat and meat product, such as the use of food additives, salts and antioxidant [2].Animal blood is considered as a useful natural red colorant source due to the content of hemoglobin in meat industry, but the ferrous of hemoglobin is unstable.
    [Show full text]
  • Significance of Heme and Heme Degradation in the Pathogenesis Of
    International Journal of Molecular Sciences Review Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders Stefan W. Ryter Proterris, Inc., Boston, MA 02118, USA; [email protected] Abstract: The heme molecule serves as an essential prosthetic group for oxygen transport and storage proteins, as well for cellular metabolic enzyme activities, including those involved in mitochondrial respiration, xenobiotic metabolism, and antioxidant responses. Dysfunction in both heme synthesis and degradation pathways can promote human disease. Heme is a pro-oxidant via iron catalysis that can induce cytotoxicity and injury to the vascular endothelium. Additionally, heme can modulate inflammatory and immune system functions. Thus, the synthesis, utilization and turnover of heme are by necessity tightly regulated. The microsomal heme oxygenase (HO) system degrades heme to carbon monoxide (CO), iron, and biliverdin-IXα, that latter which is converted to bilirubin-IXα by biliverdin reductase. Heme degradation by heme oxygenase-1 (HO-1) is linked to cytoprotection via heme removal, as well as by activity-dependent end-product generation (i.e., bile pigments and CO), and other potential mechanisms. Therapeutic strategies targeting the heme/HO-1 pathway, including therapeutic modulation of heme levels, elevation (or inhibition) of HO-1 protein and activity, and application of CO donor compounds or gas show potential in inflammatory conditions including sepsis and pulmonary diseases. Keywords: acute lung injury; carbon monoxide; heme; heme oxygenase; inflammation; lung dis- ease; sepsis Citation: Ryter, S.W. Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders. Int. J. Mol. 1. Introduction Sci.
    [Show full text]
  • Exploiting and Engineering Hemoproteins for Abiological
    Available online at www.sciencedirect.com ScienceDirect Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions 1 2 1 Oliver F Brandenberg , Rudi Fasan and Frances H Arnold The surge in reports of heme-dependent proteins as catalysts However, a significant hurdle is presented by the fact for abiotic, synthetically valuable carbene and nitrene transfer that a number of valuable chemical transformations, reactions dramatically illustrates the evolvability of the protein including many catalyzed by synthetic catalysts, do not world and our nascent ability to exploit that for new enzyme have biocatalytic counterparts. The necessary enzymes chemistry. We highlight the latest additions to the hemoprotein- simply do not exist. One effective approach to creating catalyzed reaction repertoire (including carbene Si–H and C–H new enzymes has been to engineer existing proteins to insertions, Doyle–Kirmse reactions, aldehyde olefinations, exhibit new, synthetically useful reactivity, mainly by azide-to-aldehyde conversions, and intermolecular nitrene exploiting their metallo-cofactors and other cofactors for C–H insertion) and show how different hemoprotein scaffolds new chemistry and improving those activities by directed offer varied reactivity and selectivity. Preparative-scale evolution [3–5]. The resulting fully genetically encoded syntheses of pharmaceutically relevant compounds catalysts could, at least in principle, be incorporated into accomplished with these new catalysts are beginning to in vivo biosynthetic pathways to access new products or demonstrate their biotechnological relevance. Insights into the provide alternative routes to existing products. Repurpos- determinants of enzyme lifetime and product yield are ing existing proteins for new chemistry, a trick used by providing generalizable cues for engineering heme-dependent evolution for eons, has proven particularly fruitful with proteins to further broaden the scope and utility of these heme-dependent proteins.
    [Show full text]