SUPEROXIDE DISMUTASE INHIBITOR SCREENING AND .
SUPEROXIDE DISMUTASE INHIBITOR SCREENING AND CHARACTERIZATIONUSING 19F NMRA thesis presented to the faculty of the Graduate School ofWestern Carolina University in partial fulfillment of therequirements for the degree of Master of Science in Chemistry.ByMegan Elizabeth ArringtonDirector: Dr. Jack SummersAssistant Professor of ChemistryDepartment of Chemistry & PhysicsCommittee Members: Dr. Jeff SchmittDr. Scott Huffman, ChemistryDr. Lori Seischab, BiologyMarch 2010
ACKNOWLEDGEMENTSI would like to thank my committee members and my director for their assistance.In particular, Dr. Jack Summers for giving me the opportunity to work on this project andfor his guidance and patience in its completion; Dr. Jeff Schmitt, for his hard work andassistance in docking calculations; and Dr. Lori Seischab and Emily Jellen-McCulloughfor their continued support. I express thanks to the research group members whocontributed to this project: Jonathan Markley, Michelle Yost, Jessica Parris, BenHickman, Amanda Nance and Corey Harrington.Many people assisted in this project and I appreciate their hard work. Dr. ScottHuffman calculated pKa values for galangin and assisted in UV-Vis Spectroscopy. Dr.Carmen Huffman assisted in Spartan '04 calculations. Dr. David Evanoff of WesternCarolina University and Michael Samuel of Wake Forest University analyzed MassSpectrometry samples. Brian Sneed and Dr. Brian Dinkelmeyer purified chalcones.Amy Freund of the Bruker BioSpin Corporation performed NMR high throughputscreening. Thank you again for all you have provided to this project.I also would like to thank Dr. Jeff Schmitt and Bent Creek Institute for the use ofcomputational software and Dr. Lori Seischab for the assistance and use of UV-Visspectrometer microplate reader. I appreciate the compounds provided by Dr. WilliamKwochka and Dr. Scott Huffman, and the Chemical Methodologies and LibraryDevelopment Center at the University of Pittsburgh.I would like to give special thanks to the North Carolina Biotechnology Center forthe basic research grant, which supported this project. Finally, I am grateful to thefaculty of the Department of Chemistry and Physics, Western Carolina University, andmy family for their continued encouragement in my coursework and research.
TABLE OF CONTENTSPageList of Tables . 5List of Figures . 6List of Abbreviations and Symbols . 7Abstract . 91. Introduction. 111.A. Superoxide . 111.B. Superoxide Dismutase . 111.C. Targeting Superoxide Dismutase. 121.D. SOD Assays . 131.E. 19F NMR Based Assays . 141.F. Phytoestrogen CuZnSOD Inhibition . 162. Materials and Methods . 192.A. CuZnSOD and MnSOD Standardization . 192.B. Stock Solution Preparation . 192.B.1. Sample Solution Overview . 192.B.2. Buffer Preparation . 202.B.3. Preparation of Assay Controls . 202.C. 19F NMR Based Screening . 212.D. HTS Activity Determination . 212.E. CPMG Pulse Sequence . 222.F. Enzyme Inhibition Kinetics . 232.G. pH Dependence of Inhibition Kinetics . 252.H. Aggregation Experiments . 262.I. High Throughput Screening . 272.I.1. Compound Preparation. 272.I.2. Bruker Sample Preparation and CuZnSOD Screening. 272.I.3. CuZnSOD Rescreening and MnSOD Screening . 282.J. Selective Inhibitor Screening . 282.J.1. Flavonol Screening . 282.J.2. Benzoic Acid, Diketone, and Chalcone Screening . 292.J.3. Acetophenone Screening . 292.K. Flavonol pKa Determination . 302.L. Mass Spectrometry of Quercetin Inhibited CuZnSOD . 302.M. Docking Calculations. 312.M.1. eHiTS Docking Calculations . 312.M.2. Ligand Preparation . 322.M.3. Receptor File Preparation . 332.M.4. AutoDock Analysis . 343. Results and Discussion . 363.A. Flavonol Inhibitor Screening . 363.A.1. Flavonol Observed Rate Constants . 363.A.2. Arginine Modification . 373.A.3. 2,4-Pentanedione and Glyoxal Screening . 383.A.4. Diketone Tautomerization . 383.B. pH Dependence of CuZnSOD Inhibition . 39
3.B.1. Acidic Inhibitor Screening . 393.B.2. Determination of Enzyme Acid Dissociation Constant . 403.B.3. Possible Enzymatic Causes of pH Dependence . 413.B.4. Replication of Enzyme Acid Dissociation Constant. 423.B.5. Acetophenone Screening . 423.C. Enzyme Kinetics . 443.C.1. Kinetic Data Indicate Rapid Pre-Equilibrium . 443.C.2. Effect of pH on Initial Activity . 453.C.3. Effect of Flavonol Protonation State on Dissociation Constant . 463.D. pH Dependence of CuZnSOD Inhibition Considerations . 473.E. Myricetin and Kaempferol Aggregation . 473.F. Determination of Flavonol Acid Dissociation Constants . 503.G. High Throughput Screening. 513.H. Quercetin Inhibition of CuZnSOD Observed by Mass Spectrometry . 523.I. Docking Calculations. 543.I.1. Global Analysis . 543.I.2. Analysis of Specific Ligands . 573.I.2.a. Myricetin . 573.I.2.b. Quercetin . 583.I.2.c. Taxifolin. 583.I.2.d. Kaempferol . 593.I.2.e. Apigenin . 603.I.3. Overall Docking Analysis . 603.I.4. Structure Activity Relationship . 624. Conclusions . 64References . 66Appendix . 69A. Chalcone Structures Screened for MnSOD Inhibition . 69B. Dissociation Constants for CuZnSOD Inhibitors at pH 8 . 70C. General Flavonol Structure . 70D. Flavonols Screened During the Course of this Project . 71
LIST OF TABLESTablePage1. Buffers Used in Sample Preparation at Various pH Values . 202. Compounds Screened in Inhibition of CuZnSOD at 1 mM and pH 10.59 . 293. Observed Rate Constants for Various Flavonols at pH 10.59 . 364. Dissociation Constants for Myricetin and Morin . 465. Estimated Dimerization and Dissociation Constants for Myricetin and Morin . 506. Flavonol pKa Values Determined by UV-Vis Spectroscopy . 50
LIST OF FIGURESFigurePage1. Effect of SOD on 19F Spectra . 152. Ratio of tfa to fluoride 19F Resonance Integrals Correlate with SODConcentration . 153. General Flavonol Structure . 174. Tautomers of Quercetin . 325. Reaction of 2,4-Pentanedione with Arginine forms N-substituted-2-amino4,6-dimethylpyrimidine . 376. Effect of pH on the Apparent Rate Constant of CuZnSOD Inhibition by1 mM 9-Anthracenecarboxylic acid . 417. Structure of 2,4,6-Trihydroxyacetophenone as Compared to theFlavonoid Structure . 428. Inhibition of CuZnSOD by THAP at pH 8.58 . 439. Structure of 4-hydroxyacetophenone . 4310. CuZnSOD Inhibition by 1 mM ACA at pH 8.58 . 4411. Concentration Dependence of Initial Activity Indicates an EquilibriumReaction with a Dissociation Constant Kd of 1 mM for ACA. 4512. Effect of pH on the Initial Activity of CuZnSOD Inhibition by 1 mM ACA . 4513. Initial Activity Changes Non-linearly with Myricetin Concentration at pH 7.94. 4714. Beer's Law Plot for Kaempferol at pH 7.94. 4815. Concentration Dependence of Initial Activity and Absorbance forMyricetin at pH 8 . 4916. Mass Spectra of CuZnSOD Samples . 5217. Observed Trends in Calculated Binding Energy . 5518. Kaempferol Enol Species Comparison . 6019. Poses with Arg143 and 3-oxygen Interactions Correlate to Experimental Data . 61
LIST OF ABBREVIATIONS AND SYMBOLS2ME . 2-Methoxyestradiol4HAP/di-OH. 4-HydroxyacetophenoneA/A0/%SODAc. Activity/ Initial Activity/ Percent ActivityAbs . AbsorbanceACA . 9-Anthracenecarboxylic acidArg . ArginineC1/C2 . Constants based on fluoride resonance sensitivity torelaxation by the enzyme, the delay, and F- and tfaconcentrationCPMG . Carr-Purcell-Meiboom-GillCuZnSOD. Copper-Zinc Superoxide DismutaseDHCH . SO . DimethylsulfoxideeHiTS . Electronic High Throughput ScreeningFeSOD . Iron Superoxide DismutaseHEPES . N-[2-Hydroxyethyl]piperazine-N'-[2-Ethanesulfonic acid]HTS . High Throughput ScreeningI/I0 . NMR Resonance Integral/ Initial Resonance IntegralIC50 . Half Maximal Inhibitory ConcentrationInh . Inhibitor (Monomer)Inh2 . Inhibition DimerInhTot . Total/Experimental Inhibitor Concentrationk . Rate Constant for R2 NMR Relaxation RateK1 . Association Binding Constantk2 . Rate Constant for Inhibition Step 2kapp . Apparent Rate ConstantKd/Kd,max . Dissociation Constant/ Experimental Dissociation ConstantKdim . Dimerization Constantkobs. Observed Rate ConstantKaemp . KaempferolLys . LysineMnSOD . Manganese Superoxide DismutaseMyr . MyricetinNADH . Nicotinamide Adenine DinucleotideNBT . Nitroblue TetrazoliumPIPES. Piperazine-N,N'-bis(2-Ethanesulfonic Acid)PMS . Phenazine MethosulfateR2 . NMR Transverse Relaxation Rate%SODInAc . Percent of Inactive SODSOD . Superoxide Dismutaseτ. Delay Timet . TimeT1. Spin Lattice Relaxation Rate ConstantT2. Spin-Spin Relaxation Rate ConstantTEA . Triethylaminetfa . Trifluoroacetate
THAP/tri-OH . 2,4,6-TrihydroxyacetophenoneThr. ThreonineTris . Tris(hydroxymethyl)aminomethane
ABSTRACTSUPEROXIDE DISMUTASE INHIBITOR SCREENING AND CHARACTERIZATIONUSING 19F NMRMegan Elizabeth Arrington, M.S.Western Carolina University (March 2010)Director: Jack Summers, Ph.D.Superoxide dismutase enzymes (SOD) catalyze the disproportionation of superoxide toform molecular oxygen and hydrogen peroxide in a cyclic mechanism. SODs preventthe formation of hydroxyl radicals, preventing apoptosis. Up-regulation of this enzymeimplicates it in the survival of cancer cells and pathogenic bacteria, leading to a call forSOD inhibitors as potential drugs.19F NMR based assays were used to study inhibitionof CuZnSOD by flavonol compounds. Flavonols are more effective inhibitors at high pH.We hypothesize that inhibition at high pH occurs through a two-step reaction, where thefirst step is an equilibrium reaction affected by the deprotonation of the inhibitor and thesecond step is slower and affected by the deprotonation of the enzyme. The pHdependence of the second inhibition step is consistent with enzyme deprotonation ofactive site Lys 122 at pH 10.1 and is not necessary for the first step. It was alsoobserved that aggregation of flavonol inhibitors may be occurring and therefore flavonolbinding is stronger than experimentally measured.To understand the factors that affect binding of flavonols to CuZnSOD, AutoDock4.0 calculations were carried out and compared to experimental binding constants at pH8. Experimental binding data show that flavonol diketone tautomerization is notnecessary for binding and that inhibitors bind more effectively above their pKa1 values.
Bis-deprotonated, enol and S-diketone tautomers were predicted to bind CuZnSODpreferentially in docking results. Computational results indicate that lysine and arginineresidues contribute significantly to binding by hydrogen bonding with flavonol 3,7, and 4'oxygens. Despite a strong overall correlation between docking scores for thedeprotonated species and experimental results, apigenin was predicted to have a higherbinding affinity than is experimentally observed. The underlying cause of thisdiscrepancy is a matter of further investigation.
111. INTRODUCTION1.A. SuperoxideSuperoxide is a radical anion produced by metabolic pathways in the bodythrough the reduction of molecular oxygen.1After it is produced, superoxide can reactwith other biological molecules by either univalent oxidation or reduction.2 It may alsoform the hydroxyl radical through the Fenton reaction, where superoxide acts byreducing iron as shown in Equation 1A and 1B.3O2. Fe3 O2 Fe 2 (1A)H 2O2 Fe2 .OH OH Fe3 (1B)The hydroxyl radical is a reactive oxygen species, which can modify nucleic acids andproteins through oxidation. The formation of reactive oxygen species by superoxideleads to oxidative stress.11.B. Superoxide DismutaseSuperoxide is a natural product of aerobic metabolism and is often produced inlarge quantities (107 radicals per day in rat mitochondria)1 therefore; a reaction ofsuperoxide must be present to protect the cell from damage.4 The superoxidedismutation reaction was first proposed by McCord and Fridovich4 and is shown inEquation 2.O2. O2. 2 H O2 H 2O2(2)In the dismutation reaction, one superoxide molecule is oxidized and the other is4reduced resulting in the formation of molecular oxygen and hydrogen peroxide. Theoverall reaction of superoxide is catalyzed by the enzyme superoxide dismutase.
12Superoxide dismutases (SODs) contain metal cofactors, which catalyze the reaction asshown below in Equation 3A and 3B.5M n O2. M ( n 1) O2(3A)M ( n 1) O2. 2 H M n H 2O2(3B)The reaction of superoxide catalyzed by SOD prevents oxidative stress, as superoxidewould otherwise form the hydroxyl radical, which is cytotoxic.2 The SOD reaction has arate constant of 5 x 1011 M-1s-1, showing that this enzyme has extremely high activity,making it extremely efficient at converting superoxide to hydrogen peroxide andimportant to cell survival.61.C. Targeting Superoxide DismutaseSuperoxide is generated by antibiotics to induce oxidative stress in bacterialcells. Kohanski et al. showed that bactericidal antibiotics kill bacterial cells through theproduction of hydroxyl radials from superoxide via the Fenton Reaction.7 One possibleway to increase antibiotic efficacy is to interfere in the processes that protect cells fromsuperoxide and the hydroxyl radical.7 Inhibiting superoxide dismutase would increasethe bacteria's sensitivity to these reactive oxygen species,8 making it easier to kill thecell. SOD is critical to oxidation-reduction potential balance and is implicated in manydiseases from cancers8 and Amyotrophic Lateral Sclerosis9 to malaria10 andtuberculosis.7 For this reason, there has been a call for the development of SODinhibitors as drugs.Recent research shows that superoxide dismutase inhibitors have potential asdrugs. In 2000, Huang et al. examined the use of oestrogen derivatives as inhibitors ofSOD in leukemia cells.8 They determined that 2-methoxyestradiol selectively inhibited
13human SOD, killing leukemia cells while preserving the healthy lymphocytes. Theinhibition of SODs resulted in free-radical damage of mitochrondial membranes,releasing cytochrome c and causing apoptosis.8 This result shows that one can inhibitSOD, cause oxidative stress and ultimately kill cells selectively. Other known inhibitorsare metal chelating agents11 such as diethyldithiocarbamate12 and small anions such asazide, cyanide and hydroxide that compete for the enzyme's active site.8 Theseinhibitors are poor drug candidates as they are non-specific and potentially cytotoxic.81.D. SOD AssaysHigh throughput SOD assays have to be used to screen libraries of molecules forinhibition. Using the natural substrate, superoxide, as a model for selecting inhibitors isnot a possibility.10 Traditionally, the screening for SOD targets has involved the use ofSOD assays, which observe superoxide reactions.13 SOD assays involve the generationof superoxide from an enzymatic (xanthine oxidase) or non-enzymatic (NADH/PMSsystem) source. The generated superoxide reduces a detector molecule (NBT orferricytochrome c) and the absorbance is measured.13 Assays, which use an enzymaticsource of superoxide, may be inaccurate for high throughput screening (HTS) asmolecules could inhibit the superoxide source.10 Soulere discovered that SOD assaysthat generate superoxide are not selective enough for HTS because screeningcompounds can affect the redox balance of the assay.10 Compounds, which interferedwith the assay, showed either apparent inhibition or 'superoxide dismutase-like' activitysuggesting that superoxide reacted with library compounds.10 This result shows thatassays that do not involve superoxide generation must be used for accurate highthroughput screening.
141.E. 19F NMR Based Assays19F NMR assays use the fluoride anion as a superoxide mimic, where an inhibitorwould compete with fluoride for the active site.14 Viglino, et al. established the use of 19FNMR as a CuZnSOD assay.14 They measured the T1 and T2 relaxation rate constants of19 -F as a function of oxidized CuZnSOD concentration and determined that fluoridecoordinates to the copper and that the rate of binding controls the T2 relaxation rate offluoride.14 The relaxation of fluoride is so sensitive to the presence of CuZnSOD that 108M concentrations can be detected.14 Compounds that inhibit CuZnSOD (azide andcyanide) were also found to inhibit the relaxation of fluoride.14 This shows thatrelaxation can be used as an indirect detector of inhibition and thus be used forcompound screening.The 19F NMR assay was further developed by Summers15 for high throughputscreening of superoxide dismutase inhibitors. The relaxation rate of fluoride is sensitive14to the presence of oxidized SOD ; therefore, 100 catalytic units per 600 µL sample canbe detected with a low field strength spectrometer.15 As shown in Figure 1A and 1B, theresonance of the internal reference, trifluoroacetate (tfa), is less sensitive to relaxationby the enzyme, while the presence of active superoxide dismutase greatly diminishesthe peak intensity of the fluoride anion.15
15Figure 1. Effect of SOD on 19F spectra. A. 1 transient CPMG spectrum of diamagnetictfa (5 mM) and F (20 mM NaF) solution (40 ms relaxation). B. Effect ofo 100 unitsCu/Zn SOD.Trifluoroacetate is consistently less affected by the presence of active SOD whencompared to the fluoride anion. The application of pulse delay and refocusing allowstime for the F- signal to relax, enhancing of the differences between the integrals.16 Thenatural logarithm of the ratio of tfa and F- integrals [ln(Itfa/IF-)] is directly proportional to theconcentration of the enzyme as shown in Figure 2.15Figure 2. Ratio of tfa to fluoride 19F Resonance Integrals Correlate with SODConcentration.15The concentration of active enzyme [[SODAc] can be calculated from a singlemeasurement using Equation 4.
16I[ SODAc ] C1 ln I tfa C2F(4)In which Itfa/IF- is the ratio of resonance integrals and the constants (C1 and C2) arebased on the resonances' sensitivity to relaxation by the enzyme, the delay andconcentrations of F- and tfa. A benefit of using this method is the elimination of the needfor shimming between samples as the loss in field homogeneity is indicated in theresonance loss of tfa.151.F. Phytoestrogen CuZnSOD InhibitionUsing the 19F NMR based assay, the effect of 2-methoxyestradiol (2ME) onCuZnSOD activity was measured.15 Huang, et al reported an inhibitory concentration of20 µM.8 We were unable to observe inhibition at 100 µM at neutral pH using the NMRbased assay. An alternative method described by Rigo17 using the detection ofsuperoxide had the same result. This result was possibly caused by poor solubility of 2methoxyestradiol at neutral pH. As Huang, et al.8 achieved inhibition in a 50 mMcarbonate solution; the pH was likely to be above 9.3 resulting in an increased solubilitydue to the deprotonation of the phenol proton. The experiment was repeated at pH 10.3containing 50 mM carbonate, resulting in complete inhibition.Since inhibition by 2-methoxyestradiol was not observed at neutral pH, wedecided to look for other compounds that inhibit SOD at alkaline pH. We reasoned thatthe 2ME phenol group deprotonation was important and decided to look at othercompounds containing phenols. Seven phytoestrogens18 were screened for inhibition ofCuZnSOD at 50 µM and pH 10.3. Of these quercetin was found to inhibit with an IC50 of640 nM.15 Quercetin only varies slightly in structure from the inactive compound
17apigenin c; quercetin has hydroxyl groups at R1 and R2 while apigenin c does not, theoverall structure shown in Figure 3.Figure 3. General Flavonol Structure.We noted that quercetin but not apigenin inhibits CuZnSOD. These twocompounds differ by the presence of alcohol groups at R1 and R2 in quercetin that arenot present in apigenin. This observation and the binding of 2-methoxyestradiol toCuZnSOD lead our group to hypothesize that the catechol moiety on the B ring (shownin Appendix C) of quercetin may be important to binding. To further assess the role of acatechol moiety in binding, kaempferol was screened for inhibition. It was found thatkaempferol inhibited at alkaline pH with an IC50 of 160 µM. Kaempferol was found bySharma, et al. to increase the effect of doxorubicin (a chemotherapy agent) andincreased oxidative stress causing apoptosis in glioblastoma cells.19 These findings andresearch completed by our group demonstrate the potential of flavonoids as CuZnSODinhibitors.Described in this thesis is the inhibition of CuZnSOD by flavonol compounds.We also describe our efforts to increase throughput for screening. A main goal of thisstudy was the development of a flavonol structure-activity relationship using 19F NMRkinetic studies and docking calculations. The inhibition of 19F relaxation by knownCuZnSOD inhibitors, showed the ability of this assay to screen and characterize
18inhibitors for SODs. High throughput screening was performed using a 500-compoundlibrary obtained from the Chemical Methodologies and Library Development Center atthe University of Pittsburgh. Also, additional compounds were screened for theirpotential ability to inhibit. Compounds found to inhibit CuZnSOD or MnSOD havepotential as anti-cancer8 and anti-bacterial drugs.7 Structure activity relationships canhelp further development of inhibitors with increased binding affinity and specificity.
192. MATERIALS AND METHODS2.A. CuZnSOD and MnSOD Standardization75,000 unit samples of enzymes CuZnSOD and MnSOD were obtained from MPBiomedicals LLC. Enzymes were diluted to 1mL with HPLC H2O and 2 µL enzymesolution was removed and added to 600 µL stock NaF solution. The enzyme sampleactivity was determined using the CPMG16 method (Eq. 9) and the amount of enzymeadjusted until the appropriate relaxation rate was obtained (30-100 s-1). The enzymesolution was divided into portions of approximately 3000 units (40 µL) and dehydratedusing centrifuge evaporation. Once the samples had been evaporated, they wereplaced in the freezer until needed. For inhibition studies, a portion was diluted to 200 µLwith HPLC H2O to make stock solution. 10 µL of stock CuZnSOD was added per 600 µLsample. The amount of MnSOD needed was significantly smaller than for CuZnSOD,therefore only 2-3 µL of MnSOD was needed per 600 µL NMR sample. The amount ofSOD necessary to produce an appropriate relaxation rate increases with pH, and
Association Binding Constant k2. Rate Constant for Inhibition Step 2 kapp. Apparent Rate Constant Kd/K d,max. Dissociation Constant/ Experimental Dissociation Constant
Pengaruh α-Tokoferol Terhadap Profil Superoksida Dismutase dan Malondialdehida pada Jaringan Hati Tikus di Bawah Kondisi Stres THE EFFECT OF α-TOCOPHEROL ON THE PROFILES OF SUPEROXIDE DISMUTASE AND MALONDIALDEHYDE IN THE LIVER OF RATS UNDER STRESS CONDITION
The inhibition efficiency of inhibitor was determined by using equation 2. IE (1- K / K. o) 100 (2) where K is the corrosion rate with inhibitor and K. o. is the corrosion rate without inhibitor. The surface coverage area of inhibitor was measured by equation 3. θ (1 - K / K. o) (3)
4.4 Emerging and re-emerging infections 43 4.5 Clinically insignificant transfusion-transmissible infections 44 5 Blood screening, quarantine and release 45 5.1 Blood screening process 45 5.2 Approaches to blood screening 45 5.3 Pooling for serological assays 47 5.4 Sequential screening 47 5.5 Blood screening and diagnostic testing 48 5.6 Emergency screening 48 5.7 Screening plasma for .
kinase activation loop of PIK3CA and Akt inhibitor resensitized these cells to MEK inhibitor. These results demonstrate that the overactivation of Akt plays a critical role in MEK inhibitor primary and acquired resistance and implicate combined Akt/MEK inhibition as a potentially useful treatment for RAS/BRAF-mutated colorectal cancer.
b. Determine the type of inhibition and the dissociation constant, K I, for inhibitor binding to the enzyme, for the two experiments that contain an inhibitor. Inhibitor A is an uncompetitive inhibitor. We know this because both KM and Vmax are decreased by the same amount, w
If the corrosion inhibitor can take reaction with these treatment agents and give a precipitate or the other products, it can influencing the performance of the corrosion inhibitor directly, thus may affecting the e- d velopment schedule of the oil field. Use the bactericide TQ -1 that usually used in oil field, scale inhibitor HEDP,
The corrosion rate increased with increase in temperature and decreased with increase in concentration of inhibitor compared to blank. The adsorption of inhibitor on the mild steel surface has been found to obey Temkin's adsorption isotherm. Potentiostatic polarization results reveal that morpholine act as mixed type inhibitor.
weekend, your pet will be kept at the airport due to customs duty hours. If possible pets should arrive during weekday/daytime hours to prevent unnecessary stress for the pet or owner. Commercial Airline Transport . If flying commercially, contact the airline prior to purchasing tickets to ensure pets will actually be able to fly on the day of travel (e.g. ask about the airline’s regulations .
