DOCSLIB.ORG

Metmyoglobin (Pressure Effects on Ligand Binding/Dilatometry) GABRIEL B

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Metmyoglobin (Pressure Effects on Ligand Binding/Dilatometry) GABRIEL B Proc. Nati. Acad. Sci. USA Vol. 73, No. 12, pp. 4271-4273, December 1976 Chemistry Volume changes in binding of ligands to methemoglobin and metmyoglobin (pressure effects on ligand binding/dilatometry) GABRIEL B. OGUNMOLA*, WALTER KAUZMANNtf, AND ADAM ZIPPt * Department of Chemistry, University of Ibadan, Ibadan, Nigeria; and t Department of Chemistry, Princeton University, Princeton, New Jersey 08540 Contributed by Walter Kauzmann, September 17, 1976 ABSTRACT The volume changes for the binding of various although quite concentrated solutions are required (approxi- ligands to metmyoglobin and methemoglobin have been de- mately 20 g/100 cm3). On the other hand, hemoglobin is ad- termined from the effect of pressure on the binding constants low pressures, undergoing a (for metmyoglobin) and by direct dilatometry (for methemo- versely affected by relatively globin). The volume changes associated with the binding of transition between 1 atm (0.1 MPa) and 2000 atm (200 MPa) cyanide and azide ions to methemoglobin are pH-dependent. (7), which is just the pressure range required for the determi- The volume change for the binding reaction is evidently af- nation of the slope of the plot of In KL against pressure. fected by the same subtle structural variations that have been Metmyoglobin, on the other hand, is much more stable to judged to be present from the variation with pH of enthalpy and pressure, being quite unaffected by pressures well above 2000 entropy for the binding reactions in these proteins. Hydration atm at 6-8 and 2° (8). Therefore, it is possible to determine changes and spin state changes which have been postulated to pH be linked with structural variations in these proteins must be AV for ligand binding to metmyoglobin through the study of pH-dependent. the effect of pressure on the binding constant. The volume changes accompanying the binding of oxygen The volume changes, AV, that result from chemical reactions and ethylisocyanate by ferrohemoglobin have been studied by involving proteins are believed to be influenced by changes in Johnson and Schlegel (9) and by Suzuki et al. (10) from the conformation and hydration that accompany these reactions, effect of hydrostatic pressure on the binding constants. It was but little is known about the relationship between these volume found that AV = 0 for the binding of oxygen and AV = -23 changes and the structural changes in the protein and in the cm3/mol of ligand bound for ethylisocyanate. No studies of the solvent that are responsible for them. effect of pH on AV have as yet been reported, however. The thermodynamics of the binding reactions of different methemoglobins and of sperm whale metmyoglobin with MATERIALS AND METHODS various ligands has been investigated by studying the effect of temperature on the equilibrium constant, KL, for the reaction Human hemoglobin was prepared by the usual method and in which a ligand replaces the water molecule in the sixth oxidized to methemoglobin with 2-fold excess of potassium coordination position of the iron atom in the heme (1-4). The ferricyanide (1). Ferrocyanide and excess ferricyanide were point of interest of this reaction is that although the ligand removed by dialysis against 0.05 M Bis-Tris buffer at pH 6.0. binding reactions of all methemoglobins studied so far show a Concentrated hemoglobin solutions for the dilatometric mea- relatively small dependence of log KL (and therefore of AG) surements were prepared by extended dialysis under pressure on the pH between pH 6 and 9, AH and AS are strongly pH- against appropriate buffer solutions, so that the dialysate was dependent. It has been suggested that these large compensating in osmotic equilibrium with the electrolyte in the protein so- effects might arise in at least two ways: (a) through confor- lution. mational changes in the protein, which would give rise to en- Sperm whale myoglobin, obtained from Sigma, was used thalpy and entropy changes of the same sign, and would without further purification. Buffers used in the pressure ex- therefore compensate at least to some extent (2, 3, 5); and (b) periment were cacodylate and Tris; the Tris buffer was pre- hydration changes, which might give rise to exact compensation pared from Trizma base (Sigma) and Trizma-HCI (Sigma), if water in a hydration sheath underwent a continuous "phase which had been dried under reduced pressure prior to use. change" while always in equilibrium with the solvent water (6). Bis-Tris and Tris buffers were used in the dilatometric exper- Since large volume changes might be expected to accompany iments. either of these two mechanisms, it is of interest to determine All methemoglobin and metmyoglobin solutions studied were whether there is a large pH dependence of AV for the binding 0.05 M in buffer. All measurements were made at 25.0°. reactions of the heme proteins. High Pressure Studies. The high pressure optical bomb was Volume changes of chemical reactions can be most conve- designed by W. B. Daniels. This bomb and other features of the niently measured in two ways for systems that are of inter- high pressure experiment have been described elsewhere (8, est here: by mixing the heme protein and the ligand in a 11, 12). dilatometer, and by the effect of pressure on the binding The ionization constant, K5, of metmyoglobin was deter- constant through the thermodynamic relationship AV = mined spectrophotometrically (1) at pressures of 1, 500, 1000, -RT d In KL/dP. Typically, dilatometry requires rather large 1500, 2000, 2500, and 3000 kg/cm2; spectra were recorded at amounts of reactants (0.1 meq, or of the order of 1 g of heme each pressure for seven different solutions at pH values of 6.2, protein per measurement) so it is not suitable for use with ex- 8.2, 8.4, 8.6, 8.8, 9.0, and 12.0. Corrections to the absorbance pensive proteins such as myoglobin. Fortunately hemoglobin due to compression of the solvent were made using the P-V data is available in sufficient amounts for this not to be a problem, on water of Grindley and Lind (13). The volume change on ionization was found by plotting In KI against the pressure; t To whom requests for reprints should be sent. within the experimental error this plot was a straight line. 4271 Downloaded by guest on September 29, 2021 4272 Chemistry: Ogunmola et al. Proc. Natl. Acad. Sci. USA 73 (1976) The binding constants of azide, fluoride, formate, and im- Table 1. Volume changes for binding reactions of idazole to metmyoglobin were determined by the method de- myoglobin (Mb) from the pressure dependence of the scribed by Anusiem et al. (2). For each ligand, spectra were binding constant recorded at 1, 500, 1000, 1500, and 2000 kg/cm2, at each of 10 concentrations of the ligand. In all measurements above 1 atm, AV, corrections to the absorbance due to compression of the solvent Binding reaction pH ml/mol ligand were made as described above. Values of the binding constant, MbOH, + N- MbN3 + H20 6.0 -8.95 ± 1.8 ,KL, were calculated for each solution at each pressure. For each 7.0 -9.6 ± 3.5 solution, In KL was plotted against the pressure. The mean slope MbOH2 + F-- MbF + H20 6.0 -3.3 ± 1.0 of the straight lines drawn through each set of points was used MbOH2 + formate - Mb- to calculate AV. The error limits reported for AV are estimated formate + H20 6.0 7.5 ± 1.0 from the maximum and minimum slopes consistent with the MbOH2 + imidazole - Mb- data as a whole for each ligand. imidazole + H20 6.0 0.0 ± 2.0 Dilatometric Measurements. Volume changes were found MbOH2 + OH- - MbOH + when 5 ml of buffered methemoglobin solution were mixed in H20 8-9 11.0 ± 1.2* Carlsberg dilatometers with 1 ml of excess ligand in buffer of MbOH2 - MbOH- + H+ 8-9 -11.7 ± 1.2 the same composition as that which was in dialysis equilibrium with the methemoglobin solution during its preparatikn. The * This value was found by adding 23 ml (= AVfor the reaction H20 - H+ + OH-) to the volume change for MbOH2 - MbOH- + dilatometric techniques were those described by Linder- H+. strom-Lang and Lanz (14). For each methemoglobin solution, control experiments were run in which the methemoglobin solutions were mixed with buffer not containing the ligand. The DISCUSSION small volume changes that were observed here are presumably Variation of A V with pH. In interpreting the pH depen- caused by dilution of the concentrated methemoglobin solutions dence of the volume change on binding azide to hemoglobin as well as by readjustments of the ionization of the protein in the pH range 6-8, we must recognize that we are concerned caused by slight pH differences between the protein solution with the two reactions and the buffer. The volume changes observed in these control experiments (generally contractions of a few hundredths of a HbOH2 + L 1- HbL + H20 AV= AV1 [1] microliter) were subtracted from the volume changes obtained HbOH + L + HB+ on adding the ligand at the same pH. The result is the corrected HBL + B + H.,O AV = AV2 [2] volume change, Av. The volume changes, AV, in ml/mol of ligand bound, were calculated from the expression AV = where L is the ligand and HB+ is the acid form of the Bis-Tris 340 Av/[Hbl, where [Hb] is the methemoglobin concentration buffer, B. In the case of the binding of cyanide, since HCN has before mixing in g/100 ml of solution and Av is in microliters. (The number of moles of heme iron present in the dilatometer / is 0.05 [Hb]/17,000.) The methemoglobin concentrations were 20 determined spectrophotometrically as cyanmethemoglobin, using E540 = 10.9 mM-1 cm-1; the rnethemoglobin concen- trations were generally around 20 g/100 ml.
Load more

Recommended publications
  • 18,8 Quaternary Structure of Proteins
    570 CHAPTERt8 Amino Acids,Peptides, and Proteins 18,8Quaternary structure of proteins AIMS: Todefine the termssubunit dnd quaternarystructure. Io describethe quoternorystructure of hemoglobin.To distinguishomong oxyhemoglobin,deoxyhemoglobin, ond methemoglobin. Someproteins consist of more than one pollpeptide chain. Theseindiuid- ual chains are calledsubunits of the protein. Proteins composedof subunits In some proteins, polypeptide are said to haue quaternary structure. Many proteins have structures that chains aggregateto form contain subunits. Proteins consistingof dimers (two subunits), tetramers quaternary structures. (four subunits), and hexamers (six subunits) are fairly common. The pro- teins that comprise the individual subunits may be identical, or they may be different. Like the secondary and tertiary structures, the quaternary structure of a protein is determined by its primary structure. The pollpep- tide chains of subunits are held in place by the same forces that determine tertiary structure-hydrogen bonds, salt bridges, and sometimes disulfide bridges-except the forces are betweenthe polypeptide chains of the sub- units instead of within them. Hydrophobic aliphatic and aromatic side chains of subunits can aggregateto exclude water. Hemoglobin-the globular oxygen-transport protein of blood-is an example of a protein that has a quaternary structure. Max Perutz, also of the Medical ResearchCouncil laboratories,determined the structure of horse blood hemoglobin in 1959.Hemoglobin is a larger molecule than myoglo- bin. The hemoglobin molecule has a molar mass of 64,500.It contains about 5000 individual atoms, excluding hydrogens, in 574 amino acid residues. The quaternary structure of hemoglobin consistsof four peptide sub- units. TWo of the subunits are identical and are called the alpha subunits.
    [Show full text]
  • The Role of Methemoglobin and Carboxyhemoglobin in COVID-19: a Review
    Journal of Clinical Medicine Review The Role of Methemoglobin and Carboxyhemoglobin in COVID-19: A Review Felix Scholkmann 1,2,*, Tanja Restin 2, Marco Ferrari 3 and Valentina Quaresima 3 1 Biomedical Optics Research Laboratory, Department of Neonatology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland 2 Newborn Research Zurich, Department of Neonatology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland; [email protected] 3 Department of Life, Health and Environmental Sciences, University of L’Aquila, 67100 L’Aquila, Italy; [email protected] (M.F.); [email protected] (V.Q.) * Correspondence: [email protected]; Tel.: +41-4-4255-9326 Abstract: Following the outbreak of a novel coronavirus (SARS-CoV-2) associated with pneumonia in China (Corona Virus Disease 2019, COVID-19) at the end of 2019, the world is currently facing a global pandemic of infections with SARS-CoV-2 and cases of COVID-19. Since severely ill patients often show elevated methemoglobin (MetHb) and carboxyhemoglobin (COHb) concentrations in their blood as a marker of disease severity, we aimed to summarize the currently available published study results (case reports and cross-sectional studies) on MetHb and COHb concentrations in the blood of COVID-19 patients. To this end, a systematic literature research was performed. For the case of MetHb, seven publications were identified (five case reports and two cross-sectional studies), and for the case of COHb, three studies were found (two cross-sectional studies and one case report). The findings reported in the publications show that an increase in MetHb and COHb can happen in COVID-19 patients, especially in critically ill ones, and that MetHb and COHb can increase to dangerously high levels during the course of the disease in some patients.
    [Show full text]
  • 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]
  • Concentration of NADH-Cytochrome B5 Reductase in Erythrocytes of Normal and Methemoglobinemic Individuals Measured with a Quantitative Radioimmunoblotting Assay
    Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay. N Borgese, … , G Pietrini, S Gaetani J Clin Invest. 1987;80(5):1296-1302. https://doi.org/10.1172/JCI113205. Research Article The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity. Find the latest version: https://jci.me/113205/pdf Concentration of NADH-Cytochrome b5 Reductase in Erythrocytes of Normal and Methemoglobinemic
    [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]
  • The First Korean Family with Hemoglobin-M Milwaukee-2 Leading to Hereditary Methemoglobinemia
    Case Report Yonsei Med J 2020 Dec;61(12):1064-1067 https://doi.org/10.3349/ymj.2020.61.12.1064 pISSN: 0513-5796 · eISSN: 1976-2437 The First Korean Family with Hemoglobin-M Milwaukee-2 Leading to Hereditary Methemoglobinemia Dae Sung Kim1, Hee Jo Baek1,2, Bo Ram Kim1, Bo Ae Yoon1,2, Jun Hyung Lee3, and Hoon Kook1,2 1Department of Pediatrics, Chonnam National University Hwasun Hospital, Hwasun; 2Department of Pediatrics, Chonnam National University Medical School, Gwangju; 3Department of Laboratory Medicine, Chonnam National University Hwasun Hospital, Chonnam National University Medical School, Gwangju, Korea. Hemoglobin M (HbM) is a group of abnormal hemoglobin variants that form methemoglobin, which leads to cyanosis and he- molytic anemia. HbM-Milwaukee-2 is a rare variant caused by the point mutation CAC>TAC on codon 93 of the hemoglobin sub- unit beta (HBB) gene, resulting in the replacement of histidine by tyrosine. We here report the first Korean family with HbM-Mil- waukee-2, whose diagnosis was confirmed by gene sequencing. A high index of suspicion for this rare Hb variant is necessary in a patient presenting with cyanosis since childhood, along with methemoglobinemia and a family history of cyanosis. Key Words: ‌Hemoglobin M-Milwaukee-2, methemoglobinemia, cyanosis, congenital hemolytic anemia INTRODUCTION tion pathway.6 Methemoglobinemia occurs when metHb lev- els exceed 1.5% in blood.7 Hereditary methemoglobinemia is Hemoglobinopathy refers to abnormalities in hemoglobin often caused by methemoglobin reductase enzyme deficiency,
    [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]
  • Evidence of the Existence of a High Spin Low Spin Equilibrium in Liver Microsomal Cytochrome P450, and Its Role in the Enzymatic Mechanism* H
    CROATICA CHEMICA ACTA CCACAA 49 (2) 251-261 (1977) YU ISSN 0011-1643 CCA-996 577.15.087.8 :541.651 Conference Paper Evidence of the Existence of a High Spin Low Spin Equilibrium in Liver Microsomal Cytochrome P450, and its Role in the Enzymatic Mechanism* H. Rei:n, 0. Ristau, J. Friedrich, G.-R. Jiinig, and K. RuckpauL Department of Biocatalysis, C'entral Institute of Molecular Biology, Academy of Sciences of the GDR 1115 Berlin, GDR Received November 8, 1976 In rabbit liver microsomal cytochrome P450 a high spin (S = = 5/2) low spin (S = 1/2) equilibrium has been proved to exist by recording temperature difference spectra in the Soret and in the visible region of the absorption spectrum of solubilized cytochrome P450. In the presence of type II substrates the predominantly low spin state of cytochrome P450 is maintained, only a very small shift to lower spin is observed. Ligands of the heme iron, such as cyanide and imidazole, pr9duce a pure low spin state and therefore in the presence of these ligands no temperature difference spectra can be obtained. In the presence of type I substrate, however, the spin equilibrium is shifted to the high spin state. The extent of this shift (1) depends on specific properties of the substrate and (2) it is generally relatively small, up to about 80/o in the case of substrates investigated so far. INTRODUCTION The first step in the reaction cycle of cytochrome P450 is the binding of the substrate to the enzyme. The binding is connected with changes in the absorption spectrum especially in the Soret region from which the binding constant can be evaluated1• Moreover, in the case of the so far best known cytochrome P450 from Pseudomonas putida it has been established that at substrate binding the low spin state of the heme iron is changed into the high spin state2• In the presence of camphor, a specific substrate, bacterial cytochrome P450 exhibits in the EPR spectrum g values of 8, 4, and 1.8, typical of high spin ferric heme iron in a strong distorted rhombic field.
    [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]