Lawrence H. Piette

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Abstract Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10−5 m ) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe2+-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.

Hydroxyl radical production involved in lipid peroxidation of rat liver microsomes

Abstract The spin-traps, 5,5′-Dimethyl-1-pyrroline-1-oxide and phenyltertiary-butylnitrone have been used to investigate the primary free radical involved in lipid peroxidation. In this report, we wish to present evidence that a NADPH-dependent hydroxyl radical (OH·), which may be the primary free radical that initiates lipid peroxidation, is generated in liver microsomes. A mechanism for the generation of the hydroxyl radical in liver microsomes is also postulated.

Spin-labeling as a general method in studying antibody active site

Abstract Dimensional characteristics of the nature of the hapten combining site (HCS) for rabbit anti-DNP antibodies with various spin-labeled haptens have been determined using the method of Electron Spin Resonance and spin-labeling (1). By systematically increasing the chain length separating the hapten from the spin-label moiety, an estimate of the average “depth” of the combining site was determined to be 10 A.

Hydroxyl radical production in a purified NADPH-cytochrome c (P-450) reductase system☆

Abstract Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) we have demonstrated that hydroxyl radicals are generated indirectly from purified preparations of rat liver microsomal NADPH-cytochrome c ( P -450) reductase during NADPH oxidation. Hydroxyl radical formation is completely inhibited by p -chloromercuribenzoate, but not by metyrapone. In addition, hydroxyl radical DMPO adduct formation is blocked by added linolenic acid which, in turn, is peroxidatively degraded into malondialdehyde, suggesting that hydroxyl radicals formed from purified NADPH-cytochrome c ( P -450) reductase are capable of initiating lipid peroxidation. A mechanism for the indirect production of hydroxyl radicals from NADPH-cytochrome P -450 reductase is discussed.

Influence of flavin addition and removal on the formation of superoxide by NADPH-cytochrome P-450 reductase: A spin-trap study

Abstract The spin trap 5,5-dimethyl-1-pyrroline-1-oxide was used to measure the formation of superoxide that occurs during the oxidation of NADPH by cytochrome P -450 reductase, a flavoprotein containing one molecule each of FMN and FAD per polypeptide chain. The enzymes ability to reduce oxygen to superoxide appears to be equally dependent on its content of FMN and FAD. Selective removal of FMN from reductase decreases superoxide formation by an amount equivalent to the difference in total flavin (FMN and FAD) concentration between native and depleted preparations of the protein. In contrast, reductase-bound FMN is necessary for the transfer of electrons to another acceptor cytochrome c , but not to ferricyanide. NADPH oxidation and the concomitant formation of superoxide by native preparations of reductase are stimulated by the addition of riboflavin, FMN, and FAD with riboflavin being the most effective. The results obtained from this study suggest that added flavins are reduced by cytochrome P -450 reductase as electron acceptors and increased superoxide formation results from their autooxidation.

ESR Studies of Naphthoquinones and Related Compounds. Hyperfine Interaction by the Methoxyl Group as a Conformational Probe

ESR spectra were run on a large number of methoxy‐substituted para‐semiquinones. With the help of MO calculations it was deduced that methoxyl hyperfine interaction in these systems is, in part, a function of the spin density on the ether oxygen. By the use of model compounds it was further deduced that only one conformational isomer is effective in giving rise to splitting. A semiempirical value for |QOCH3| was calculated to be 5.6 G.

Organophosphate spin-label studies of inhibited esterases, α-chymotrypsin and cholinesterase

Abstract A new ESR spin-label has been synthesized which parallels the action of the standard DIFP (diisopropylfluorophosphate) inhibitors for esterase enzymes. The labels differ from DIFP in that one or more of the ester linkages is an organic nitroxide molecule. Preliminary inhibition studies of α-chymotrypsin, trypsin, choline-esterase, and thrombin show strikingly different spectra for the spin-labelled inhibited enzymes indicating a variation in the structure of the active site in the vicinity of the active serine. It is suggested that such labels should be of a very general use in studying the nature of the active site under catalytic conditions.

Interaction of spin-labeled analogues of 1,10-phenanthroline and lodoacetamides with Horse Liver Alcohol Dehydrogenase☆

Abstract A paramagnetic analog of 1,10-phenanthroline has been synthesized which binds to Horse Liver Alcohol Dehydrogenase forming a stoichiometric 2:1 complex. The compound, 1,10-phenanthroline-5-acetamido-2,2,5,5-tetramethylpyrrolidine-1-oxyl was formed from 5-amino-1, 10-phenanthroline and the acid chloride of 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl. The compound is competitive with NAD + with an apparent K d = 2.7 μ m . The Electron Spin Resonance absorption of the compound exhibits a strongly immobilized spectrum with a maximum splitting of 66 gauss indicating that the label is located in a highly restricted environment most likely bound to two of the four zincs present in LADH. NADH and 1,10-phenanthroline prevent binding of the paramagnetic analog to the enzyme. A low zinc (approximately two zincs remaining) and a zinc-free LADH analog do not bind the label indicating that the most easily dissociable zincs in the native enzyme are those to which the label binds and are probably catalytically important. Two sulfhydryls presumably associated with enzyme activity have been labeled with two iodoacetamide spin labels with varying chain lengths. The Electron Spin Resonance spectra obtained from these labels indicate that like zinc, these thiols also exist in restricted environments. ESR spectra, stoichiometry of binding and remaining catalytic activity of the spin-labeled LADH suggests that the two catalytically important thiols are in structurally different environments. Inhibition of LADH with the iodoacetamide spin labels reduces the stoichiometry of binding of the paramagnetic analog of 1,10-phenanthroline. These results are suggestive of a close proximity of the zinc and sulfhydryl moieties within the enzymes active site.

ESR STUDY OF ORIENTED TRIPLET MOLECULES GENERATED BY EXCITATION WITH POLARIZED LIGHT

Abstract— The lowest‐lying triplet states of a variety of aromatic molecules and complexes have been generated by the irradiation of these compounds in solvent glasses with plane‐polarized light. Measurements of the allowed (ΔM=± 1) ESR transitions clearly demonstrate that the triplets so formed are oriented with respect to the external magnetic field. By this method the triplet zero‐field splitting parameters, D and E, can be evaluated simply and reliably. Intramolecular energy transfer is postulated to explain the triplet spectra of Zn(o‐phen)2(NO3)2 and Zn(o‐phen)3(NO3)2. It was observed that in triplet‐triplet energy transfer from benzophenone to naphthalene there is no apparent orientation requirement between the donor and acceptor molecules. Further areas of significance and application of this technique are suggested.

ESR probing of macromolecules: Spin label study of the conformational integrity and transitions in modified α-chymotrypsin☆

Abstract The spin-labeling method has been used to probe the effect of specific modification of α-chymotrypsin on the conformational integrity of this enzymes active center. Labels on either the serine 195 hydroxyl or methionine 192 thioether or both provide spectral data which indicate the following: (1) alkylation of one or both of the methionines in α-chymotrypsin with all reagents tried causes a loss of “structure” of the active center; (2) conversely, oxidation of these residues has little effect; and (3) this loss of structure has little effect on the poising of the catalytically functional groups, but diminishes the productivity of substrate binding via steric inhibition. Associated with this latter point is evidence that methionine 192 moves in response to the ionization of aspartic acid 194 and isoleucine 16. The relationship of this evidence to the crystallographic model of the enzyme and its catalytic operation is discussed.