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Coronavirus disease 2019 (COVID-19), which is the pandemic caused by the virus, severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), first appearing in December 2019, continues to confound the world. In this update we provide insights into how some of the new mutant variant strains of SARS CoV-2 have evolved to be more infective. We also introduce our supplement of the special issue on the topic of the proteins of SARS CoV-2 in the Protein Journal, which follows this introduction.

Copper is one of the most abundant biological metals, and its chemical properties mean that organisms need sophisticated and multilayer mechanisms in place to maintain homoeostasis and avoid deleterious effects. Studying copper proteins requires multiple techniques, but electron paramagnetic resonance (EPR) plays a key role in understanding Cu(II) sites in proteins. When spin-labels such as aminoxyl radicals (commonly referred to as nitroxides) are introduced, then EPR becomes a powerful technique to monitor not only the coordination environment, but also to obtain structural information that is often not readily available from other techniques. This information can contribute to explaining how cuproproteins fold and misfold. The theory and practice of EPR can be daunting to the non-expert; therefore, in this mini review, we explore how nitroxide spin-labelling can be used to help the inorganic biochemist gain greater understanding of cuproprotein structure and function in vitro and how EPR imaging may help improve understanding of copper homoeostasis in vivo.

This is a historical overview of the advent of applications of spin labeling to biological systems and the subsequent developments from the perspective of a scientist who was working as a Ph.D. student when the technique was conceived and was fortunate enough to participate in its development. In addition, the historical development of in vivo applications of EPR on animals and other living systems is described from a personal perspective.

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry 2004, 43, 5575). Association of alpha-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of alpha-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of alpha-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human alpha-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of alpha-lactalbumin in a complex with poly-Lys(Arg), named alpha-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other alpha-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of alpha-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues.

The study of small Asp-Phe analogs was undertaken since this dipeptide sequence is critical in fibrinogen recognition and catalysis. The inhibition of clotting activity by Asp-Phe-methyl ester (aspartame), formyl-Asp-Phe-methyl ester and acetyl-Asp-Phe was biphasic in all cases, indicating the presence of at least two binding sites. The N-terminally blocked derivatives are stronger inhibitors than aspartame. In contrast, tosyl-Gly-Pro-Arg-p'-nitroanilide hydrolysis was inhibited minimally by Asp-Phe-methyl, ester [Ki(app)=98 mM]. Acetyl-Asp-Phe inhibition of thrombin amidase activity was biphasic, tenfold stronger and appeared to be strongly cooperative. These results are discussed with respect to the inhibition of alpha-thrombin by ATP.

The applications of in vivo electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy have been impressive over a relatively short period despite the many obstacles which had to be overcome, such as dielectric absorption and biodestruction. The loop-gap resonators and modified loop coil systems have emerged as the most suitable resonators for in vivo EPR experiments. This paper briefly discusses instrumental aspects as a prelude to several examples related to the in vivo monitoring and detection of bioradicals. Recent progress in detection of bioradicals induced by drugs or chemicals is discussed with regard to nitrosocompounds, nitric oxide and metals in vivo. A clinical EPR application is also discussed., (Copyright 2004 John Wiley & Sons, Ltd.)

The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is complexed with ferrous iron-dithiocarbamate ligand spin trap complexes. Despite the minimally invasive nature of the technique, highly sensitive localized concentrations of NO may be observed ("trapped") in vivo by both L-band EPR and magnetic resonance imaging.

Experiments using purified recombinant human NAD(P)H:quinone oxidoreductase 1 (NQO1) revealed that the auto-oxidation of fully reduced protein resulted in a 1:1 stoichiometry of oxygen consumption to NADH oxidation with the production of hydrogen peroxide. The rate of auto-oxidation of fully reduced NQO1 was markedly accelerated in the presence of superoxide (O(2)(*)(-)), whereas the addition of superoxide dismutase greatly inhibited the rate of auto-oxidation. The ability of reduced NQO1 to react with O(2)(*)(-) suggested a role for NQO1 in scavenging O(2)(*)(-), and this hypothesis was tested using established methods for O(2)(*)(-) production and detection. The addition of NQO1 in combination with NAD(P)H resulted in inhibition of dihydroethidium oxidation, pyrogallol auto-oxidation, and elimination of a potassium superoxide-generated ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide:O(2)(*)(-) adduct signal (electron spin resonance). Kinetic parameters for the reduction of O(2)(*)(-) by NQO1 were estimated using xanthine/xanthine oxidase as the source of O(2)(*)(-) and after NQO1-dependent NADH oxidation at 340 nm. The ability of NQO1 to scavenge O(2)(*)(-) was also examined using cell sonicates prepared from isogenic cell lines containing no NQO1 activity (NQO1(-)) or very high levels of NQO1 activity (NQO1(+)). We demonstrated that addition of NAD(P)H and cell sonicate from NQO1(+) but not NQO1(-) cells resulted in an increased level of O(2)(*)(-) scavenging could be inhibited by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. NQO1 can generate hydroquinones that are redox active, and the O(2)(*)(-) scavenging activity of NQO1 may allow protection against O(2)(*)(-) at the site of hydroquinone generation. In addition, the O(2)(*)(-) scavenging activity of NQO1 may provide an additional level of protection against O(2)(*)(-) induced toxicity.

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.

Alpha-lactalbumin (alpha-LA) is biosynthesized and stored at the smooth endoplasmic reticulum (ER), then transferred to the Golgi lumen when prolactin stimulation of lactose biosynthesis and secretion takes place. Because both environments are composed of membranes, it was of interest to examine the interactions of alpha-LA with relevant model and biological membranes. Using the ESR spin-labeled fatty acid analog 5-doxyl stearic acid, we found evidence reflecting the insertion of "acid-shocked" molten globule (MG) alpha-LA into lecithin or phosphatidylserine (PS) multi-lamellar vesicles. An additional approximately 3 G immobilization was observed in the alpha-LA-lecithin sample versus the lipid alone. With PS, the increased immobilization was almost 6 G, reflecting an enhanced effect caused by strong electrostatic interactions between the positively charged protein with the negatively charged headgroup at pH 2.4. This was also reflected in the broadening of the PS:alpha-LA phase transition. Additionally, we have demonstrated that alpha-LA in its apo-form also shows similar insertion characteristics with both model and natural lipid membranes. Upon addition of calcium, the apo-form is released from the membrane as the Ca(2+)-bound protein.

The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of pentylenetetrazole (PTZ)-kindling as an animal model of primary generalized epilepsy. The daily administration of PTZ is associated with an increase in the amount of neuronal nitric oxide synthase (nNOS). NO generation was measured directly by in vivo and ex vivo electron paramagnetic resonance on rodents undergoing progressive convulsions. We found that primary generalized epilepsy is caused by NO induction during the persistent up-regulation of nNOS expression, but that NO induction is not associated with severe generalized seizures following long-term kindling phenomena after PTZ withdrawal. Morphological changes in the brain structure of rats were measured by magnetic resonance imaging during epileptic convulsions induced by repetitive administration of PTZ. Cerebellum volume for kindled rats decreased 20% but not in rats treated with the nNOS inhibitor, 3Br-7NI, suggesting that generation of NO in the cerebellum is related to decrease in cerebellum volume following PTZ-kindling.

The role of nitric oxide (NO) in epileptogenesis was studied in pentylenetetrazole (PTZ)-treated animals using in vivo and ex vivo EPR spectroscopy. NO generation was measured directly in the brain of a PTZ-induced mouse in vivo by an L-band EPR spectrometer. An elevation in NO production in the brain was observed during convulsions, and more NO was generated in the tonic seizure vs. the clonic seizure. NO content in several brain tissues (including the cerebral cortex (CR), cerebellum (CL), olfactory bulb (OB), hippocampus (HI), and hypothalamus (HT)) of PTZ-doped rats was analyzed quantitatively ex vivo by X-band EPR. To test the involvement of NO in seizure development, pharmacological analyses were performed using the NO synthase (NOS) inhibitors N(G)-nitro-L-arginine (L-NNA), N(G)-monomethyl-L-arginine (L-NMMA), and 3-bromo-7-nitroindazole (3Br-7NI). All of these inhibitors suppressed the convulsions, holding them at the clonic level, and prevented development of a tonic convulsion in rats doped with up to 80 mg/kg PTZ. 3Br-7NI completely inhibited NO production, but L-NNA and L-NMMA showed only 70% inhibition of NO production in PTZ-doped rats. In order to examine the contributions of NO in convulsions, rats were treated with anticonvulsants (phenytoin and diazepam) before PTZ treatment. Both drugs completely suppressed tonic convulsion in PTZ-doped rats at doses up to 80 mg/kg, but NO levels were similar to those detected in a clonic convulsion. These results support the notion that NO does not directly induce a clonic convulsion, but may be generated as a consequence of onset of seizure., (Copyright 2002 Wiley-Liss, Inc.)

The enzymatic generation of nitric oxide (NO) in vivo has been reported to be modulated by ions, such as copper and iron. Superparamagnetic iron oxide (SPIO) or ferumoxides is a liver-specific magnetic resonance contrast agent that is taken up by the Kupffer cells, where NO is generated by inducible nitric oxide synthase (iNOS). Thus, it is important to evaluate SPIO in vivo under conditions, such as infectious disease, where significant amounts of NO are generated by iNOS. In this study, we monitored the pharmacokinetics of SPIO in the liver of septic-shock mice and rats. A significant decrease in the ferric iron EPR signal was observed during NO generation in septic-shock mice compared with control mice doped with only SPIO. These results were also confirmed in a model reaction system consisting of SPIO and the NO donor, S-nitroso-N-acetyl DL penicillamine (SNAP). We compared NO generation quantitatively in the liver of the septic-shock rats, either in the presence or absence of SPIO, and found that the presence of SPIO did not affect the NO-generating activity of NOS expressed in the liver. T2-weighted MR images of an agarose gel phantom containing different SPIO to NO donor (SNAP) ratios clearly demonstrated that the contrast enhancement by SPIO decreased with increasing NO at constant SPIO levels. The reduced contrast is most probably due to the reduction of ferric to ferrous irons, resulting in a decrease in paramagnetic relaxation of water protons. These results show that SPIO can be a versatile NO-sensitive indicator, especially employing MRI as a powerful tool to 'visualize' sites of NO generation.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution., (Copyright 2000 Wiley-Liss, Inc.)

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution., (Copyright 2000 Academic Press.)

Small milk protein alpha-lactalbumin (alpha-LA), a component of lactose synthase, is a simple model Ca(2+) binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca(2+) binding site, which binds Mg(2+), Mn(2+), Na(+), and K(+), and several distinct Zn(2+) binding sites. The binding of cations to the Ca(2+) site increases protein stability against action of heat and various denaturing agents, while the binding of Zn(2+) to the Ca(2+)-loaded protein decreases its stability. Functioning of alpha-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of alpha-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.

Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 A resolution, respectively. Previous electron spin resonance studies with these labels gave rise to a low-resolution "topography map" of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation/inhibition effect on thrombin activity and (2) a region sensitive to alpha-thrombin autoproteolytic cleavage(s) to gamma-thrombin (Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the gamma-cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies.

The effects of amino acid substitutions in the N-terminus of bovine recombinant alpha-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type alpha-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu(1)residue of the native sequence is genetically removed, leaving an N-terminal methionine in its place, shows almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65-72., (Copyright 1999 Wiley-Liss, Inc.)

Nifedipine [3,5-pyridinedicarboxylic acid, 1,4-dihydro-2, 6-dimethyl-4-(2-nitrophenyl)-dimethyl ester] is a calcium channel blocker that has been widely used as a prescription drug for patients with hypertension. After illumination by ordinary light for 24 hr, nifedipine is converted completely to its nitroso analog without further photochemical degradation. Evidence for stable, nitroxyl-like free radical generation in mice was observed 15 min after intramuscular (i.m.) or intraperitoneal (i.p.) injection of illuminated nifedipine as monitored by in vivo L-band electron paramagnetic resonance (EPR) spectrometry. This was confirmed in more detail by ex vivo measurements on excised muscle and liver tissue. The nature of these radicals was surmised by comparing the reaction of illuminated nitroso-nifedipine with polyunsaturated fatty acids. Surprisingly, identical radical spectra were detected from excised liver doped with nonilluminated nifedipine, suggesting that this drug can be enzymatically converted in vivo to its nitroso analog without the requirement for illumination. This is one of the first reports of in vivo EPR evidence for a class of unsaturated fatty acid radical conjugates resulting from the normal metabolism of a common drug. Magn Reson Med 42:691-694, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The concentration of nitric oxide (NO) was measured in the brain of septic-shock animals by electron paramagnetic resonance spectrometry (EPR). NO was spin trapped and quantitated in several regions of the brain (cortex, hippocampus, hypothalamus, cerebellum, and olfactory bulb) as well as other organs (liver, kidney, and heart) of rats induced with lipopolysaccharide (LPS) using Fe(II)/dithiocarbamate complexes containing diethyldithiocarbamate (DETC) or N-methyl-D-glucamine (MGD). The spin trap, (DETC)(2)-Fe(II), complexed NO generated in all tissues examined, but (MGD)(2)-Fe(II) complex was ineffective in detecting NO in the brain of septic-shock rats, although identical amounts of NO were detected in the liver with either spin trap. A triplet EPR spectrum of (DETC)(2)-Fe(II)-NO with a(N) = 12.8 gauss and g = 2.04 was observed in the cortex, hippocampus, hypothalamus, cerebellum, but not the olfactory bulb. The amount of NO in the brain was about 20% of that found in the liver. The (DETC)(2)-Fe(II)-NO signal in all the tissues of septic-shock rats was markedly suppressed by preadministration of the nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine (L-NMMA) or 3-bromo-7-nitroindazole, suggesting that the NO detected from brain tissue was produced enzymatically by NOS. In contrast to previous studies on the liver and other organs, phenyl-N-tert-butyl nitrone (PBN), did not suppress iNOS expression in brain tissue of LPS-treated rats. This could be due to a totally different regulation system for iNOS in liver versus brain tissue. Magn Reson Med 42:599-602, 1999., (Copyright 1999 Wiley-Liss, Inc.)

This study employs (31)P-nuclear magnetic resonance (NMR) to probe for changes in molecular structure arising from reactions between free radicals and a phosphorus-containing nitrone spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). A number of biologically relevant free radical reactions were detected: a) reactions of DEPMPO with ( small middle dot)OH resulted in a new (31)P-NMR resonance at 27.05 ppm (shifted from the parent compound at 23.67 ppm); evidence suggests that this species is a diamagnetic hydroxy-pyrrolidone reduction product; b) (31)P-NMR spectra of DEPMPO/( small middle dot)CH(3) reactions resulted in peaks at 24.54, 30.83, and 32.31 ppm, while DEPMPO/( small middle dot)CH(2)OH produced peaks at 24.05, 30.80 and 32.52 ppm; in the presence of excess ascorbate, only resonances between 30 and 32 ppm were evident, which we have tentatively assigned to the hydroxylamine isomers of their respective adducts; and c) reaction of DEPMPO with O(2)( small middle dot-), produced by xanthine/xanthine oxidase or stimulated neutrophils, resulted in a single line, indistinguishable from DEPMPO/( small middle dot)OH reaction products. We conclude that NMR spin trapping is a useful approach for detecting free radical reaction pathways. It may have future applications for human free radical biology and imaging. Magn Reson Med 42:228-234, 1999., (Copyright 1999 Wiley-Liss, Inc.)

This paper reports the first in vivo NMR image of the distribution of NO using the "MRI spin-trapping" technique. NO was complexed with the Fe(II)-chelate spin trap, N-methyl-D-glucamine dithiocarbamate (MGD), verified as (MGD)(2)-Fe(II)-NO by EPR, and the radical distribution was "visualized" by MR images. In rats, the (MGD)(2)-Fe(II)-NO complex was concentrated in the liver displaying significantly enhanced contrast in the vascular structure such as hepatic vein and inferior vena cava. Nitric oxide synthase was verified as the source of NO in rats with septic shock by pre-administration of the competitive inhibitor N-monomethyl-L-arginine, resulting in reduced enhancement. The NO complex was more stable in vivo and a more effective MRI contrast agent than other stable nitrogen containing radicals, such as nitroxides. The MRI spin-trapping method should be a powerful tool for visualizing spatial distributions of free radicals in pathologic organs and tissues when combined with the appropriate radical complexing agent, such as (MGD)(2)-Fe(II) used in these studies. Magn Reson Med 42:235-239, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The expression, folding, and characterization of a series of small proteins with increasingly complex disulfide bond patterns were characterized. A phagemid was prepared from the pT7-7 plasmid to facilitate mutagenic studies with these proteins. cDNAs coding for bovine, rat, and human prolactin; human growth hormone; and bovine alpha-lactalbumin were amplified by PCR using primers that inserted restriction sites at the 5' and 3' ends and reduced the coding sequence to the mature methionyl protein with bacterially preferred codons in the 5' region. The expressed proteins were folded and oxidized by methods that allowed disulfide bond formation to occur either during or following folding. The effectiveness of the folding procedures was determined for each protein by electrophoresis, absorption spectroscopy, and functional studies. The redox conditions required for folding functional proteins varied as the number of disulfide bonds per unit molecular weight increased. Human growth hormone, 22 kDa; human prolactin, 23 kDa; and bovine prolactin, 23 kDa, contain two, three, and three disulfides, respectively, and are folded correctly by air oxidation performed during renaturation under alkaline conditions. Proper disulfide bond formation of rat prolactin, 23 kDa, containing three disulfide bonds required the addition of a reducing agent at the initiation of renaturation. Bovine alpha-lactalbumin, 14 kDa with four disulfide bonds, required complete renaturation prior to the removal of a reducing agent. SDS-gel electrophoresis under nonreducing conditions provided information regarding the proper folding of these proteins. The absorption of 250-nm light by disulfide bonds also provided information regarding the proper folding of rat prolactin and bovine alpha-lactalbumin., (Copyright 1999 Academic Press.)

This paper discusses, compares and evaluates various in vivo EPR methods of detection of nitric oxide (NO). In particular the various classes of agents are: Fe(II)-dithiocarbamate derivative complexes of MGD (N-methyl-D-glucamine dithiocarbamate) and DTCS [N-(dithiocarboxy)sarcosine], stable imidazolineoxy N-oxides and nitronyl N-oxides, and NO-sensitive chars. As a specific example direct, real-time, in vivo measurements of nitric oxide (NO) in mice are described with the water soluble metal chelator complex (MGD)2-Fe(II), as monitored at L-band EPR. The three-line EPR spectrum of [(MGD)2-Fe(II)-NO] was observed non-invasively in both control animals injected with the preformed product [(MGD)2-Fe(II)-NO] and from lipopolysaccharide (LPS) treated mice subsequently injected with (MGD)2-Fe(II) complex. The [(MGD)2-Fe(II)-NO] spectrum was markedly suppressed after administration of phenyl N-tert-butyl nitrone (PBN) prior to LPS injection as PBN inhibits the expression of inducible nitric oxide synthase (iNOS). When 15N-arginine was administered to LPS-treated mice, an EPR spectrum consisting of both three- and two-line EPR signals (due to (MGD)2-Fe(II)-14NO and (MGD)2-Fe(II)-15NO respectively) was observed, confirming that the trapped NO was generated through the NOS enzyme and not other chemical routes.