Cytochrome aa3 from the aerobic photoheterotroph Erythrobacter longus: purification, and enzymatic and molecular features

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Erythrobacter longus to homogeneity as judged by polyacrylamide gel electrophoresis, and some of its properties were studied. The spectral properties of the oxidase closely resembled those of mitochondrial and other bacterial cytochromes aa3. The enzyme showed absorption peaks at 430 and 598 nm in the oxidized form, and at 444 and 603 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 600 nm. The enzyme oxidized eukaryotic ferrocytochromes C more rapidly than E. longus ferrocytochrome c. The reactions catalyzed by the enzyme were 50% inhibited by 0.7 microM KCN. The enzyme contained 1 g atom of copper and 1 g atom of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two identical subunits, each with a molecular weight of 43,000.     

Estrogen receptor stereochemistry: ligand binding and hormonal responsiveness

Estrogen stimulation of the uterus produces a spectrum of biochemical responses that are customarily linked together. This report is an overview of a series of studies by our laboratory investigating the role of different ligand structures in eliciting hormonal responses. Diethylstilbestrol (DES) and certain structural analogs, indenestrol A (IA), indenestrol B (IB), and pseudo-DES, were used as probes to segregate various genomic responses previously considered interrelated, most notably the events of specific protein synthesis and DNA synthesis. These compounds have weak uterotrophic activity; however, they interact with high affinity specifically with mouse uterine estrogen receptors (ERs). All of them produce stoichiometrically similar amounts of ER complex in the nucleus. Indenestrol A and IB possess a single chiral carbon atom and exist as a mixture of enantiomers (ENTs). Competitive binding assays of pure ENTs and cytosolic ERs demonstrated a stereochemical chiral preference for the IA isomer but not IB. This preference was also evident from nuclear ER occupancy experiments. Biologic activity of the IA ENTs also demonstrated differences as seen by receptor binding. Ornithine decarboxylase (ODC) activity was stimulated 600% by DES and partially by IA (rac). All of the ODC activity produced by IA (rac) was due to the IA(C3)-S ENT. Uterine DNA synthesis was measured after treatment with the IA compounds. Indenestrol A (rac) increased DNA synthesis to 40% of the level seen with DES. The weak ENTs showed no activity and the active ENTs were weaker than the IA racemic. These compounds should be useful probes for studying the individual responses involved in estrogen-induced uterine growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

SuperStar: improved knowledge-based interaction fields for protein binding sites

SuperStar is an empirical method for identifying interaction sites in proteins, based entirely on experimental information about non-bonded interactions occurring in small-molecule crystal structures, taken from the IsoStar database. We describe recent modifications and additions to SuperStar, validating the results on a test set of 122 X-ray structures of protein-ligand complexes. In this validation, propensity maps are generated for all the binding sites of these proteins, using four different probes: a charged NH(+)(3) nitrogen atom, a carbonyl oxygen atom, a hydroxyl oxygen atom and a methyl carbon atom. Next, the maps are compared with the experimentally observed positions of ligand atoms of these types. A peak-searching algorithm is introduced that highlights potential interaction hot spots. For the three hydrogen-bonding probes - NH(+)(3) nitrogen atom, carbonyl oxygen atom and hydroxyl oxygen atom - the average distance from the ligand atom to the nearest SuperStar peak is 1.0-1.2 A (0.8-1.0 A for solvent-inaccessible ligand atoms). For the methyl carbon atom probe, this distance is about 1.5 A, probably because interactions to methyl groups are much less directional.The most important addition to SuperStar is the enabling of propensity maps around metal centres - Ca(2+), Mg(2+) and Zn(2+) - in protein binding sites. The results are validated on a test set of 24 protein-ligand complexes that have a metal ion in their binding site. Coordination geometries are derived automatically, using only the protein atoms that coordinate to the metal ion. The correct coordination geometry is derived in approximately 75 % of the cases. If the derived geometry is assumed during the SuperStar calculation, the average distance from a ligand atom coordinating to the metal ion to the nearest peak in the propensity map for an oxygen probe is 0.87(7) A. If the correct coordination geometry is imposed, this distance reduces to 0.59(7)A. This indicates that the SuperStar predictions around metal-binding sites are at least as good as those around other protein groups. Using clustering techniques, a non-redundant set of probes is selected from the set of probes available in the IsoStar database. The performance in SuperStar of all these probes is tested on the test set of protein-ligand complexes. With the exception of the "ether oxygen" probe and the "any NH(+)" probe, all new probes perform as well as the four probes introduced first.     

Photosynthetic action spectra and adaptation to spectral light distribution in a benthic cyanobacterial mat

We studied adaptation to spectral light distribution in undisturbed benthic communities of cyanobacterial mats growing in hypersaline ponds at Guerrero Negro, Baja California, Mexico. Microscale measurements of oxygen photosynthesis and action spectra were performed with microelectrodes; spectral radiance was measured with fiber-optic microprobes. The spatial resolution of all measurements was 0.1 mm, and the spectral resolution was 10 to 15 nm. Light attenuation spectra showed absorption predominantly by chlorophyll a (Chl a) (430 and 670 nm), phycocyanin (620 nm), and carotenoids (440 to 500 nm). Blue light (450 nm) was attenuated 10-fold more strongly than red light (600 nm). The action spectra of the surface film of diatoms accordingly showed activity over the whole spectrum, with maxima for Chl a and carotenoids. The underlying dense Microcoleus population showed almost exclusively activity dependent upon light harvesting by phycobilins at 550 to 660 nm. Maximum activity was at 580 and 650 nm, indicating absorption by phycoerythrin and phycocyanin as well as by allophycocyanin. Very little Chl a-dependent activity could be detected in the cyanobacterial action spectrum, even with additional 600-nm light to excite photosystem II. The depth distribution of photosynthesis showed detectable activity down to a depth of 0.8 to 2.5 mm, where the downwelling radiant flux at 600 nm was reduced to 0.2 to 0.6% of the surface flux.     

Photosynthetic action spectra and adaptation to spectral light distribution in a benthic cyanobacterial mat.

We studied adaptation to spectral light distribution in undisturbed benthic communities of cyanobacterial mats growing in hypersaline ponds at Guerrero Negro, Baja California, Mexico. Microscale measurements of oxygen photosynthesis and action spectra were performed with microelectrodes; spectral radiance was measured with fiber-optic microprobes. The spatial resolution of all measurements was 0.1 mm, and the spectral resolution was 10 to 15 nm. Light attenuation spectra showed absorption predominantly by chlorophyll a (Chl a) (430 and 670 nm), phycocyanin (620 nm), and carotenoids (440 to 500 nm). Blue light (450 nm) was attenuated 10-fold more strongly than red light (600 nm). The action spectra of the surface film of diatoms accordingly showed activity over the whole spectrum, with maxima for Chl a and carotenoids. The underlying dense Microcoleus population showed almost exclusively activity dependent upon light harvesting by phycobilins at 550 to 660 nm. Maximum activity was at 580 and 650 nm, indicating absorption by phycoerythrin and phycocyanin as well as by allophycocyanin. Very little Chl a-dependent activity could be detected in the cyanobacterial action spectrum, even with additional 600-nm light to excite photosystem II. The depth distribution of photosynthesis showed detectable activity down to a depth of 0.8 to 2.5 mm, where the downwelling radiant flux at 600 nm was reduced to 0.2 to 0.6% of the surface flux.     

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.     

Identification of cytochrome a and a3 in yeast cells.

1) Cells of Saccharomyces cerevisiae have been analysed by single and double-bean spectroscopy. Evidence is given for two components of cytochrome c oxidase in the alpha-region of their absorption spectrum. A rapidly reduceable component with a maximum at 600 nm and a slowly reduceable component with a maximum at 604 nm contribute about equal amounts to the total alpha-absorption of cytochrome c oxidase. 2) The component absorbing at 600 nm was identified as the high-potential component with a redox potential of 340 - 355mV, and the 604-nm component as the low-potential component of cytochrome c oxidase with redox potential of 180 - 190 mV. 3) Both components can be characterized by analysing the reduction kinetics in the presence of carbon monoxide. In the presence of saturating concentrations of carbon monoxide, an oxygen pulse leads to a rapid oxidation and subsequent reduction of cytochrome c oxidase, but the rapid reduction phase at 600 nm completely disappears, demonstrating its identity with cytochrome a3, which, being liganded by carbon monoxide in its reduced state, cannot react any more. The component which becomes oxidized and later reduced in the presence of carbon monoxide -- by definition cytochrome a -- has an absorption maximum at 604 nm. 4) The total extinction change at 604 nm in the presence of carbon monoxide is nearly as high as in its absence, but the reduction occurs in two phases and only the second phase, which contributes 50 - 60% to the total absorbance, corresponds in redox potential and kinetic properties to cytochrome a. Because the redox potential of the first reduction phase is very close to that of the low-potential copper atom of cytochrome c oxidase, it is concluded that the apparent increase in the extinction coefficient of cytochrome a in the presence of carbon monoxide is the result of a strong interaction between the ligand fields of cytochrome a and copper, induced by the binding of carbon monoxide to reduced cytochrome a3.     

Secretory expression of thermostable T1 lipase through bacteriocin release protein

The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A(600 nm), temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 microM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A(600 nm) of 1.25, 30 degrees C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host.     

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.     

The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coliα-hemolysin family

Abstract Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype. In this study the hemolytic activity of E. coli O157:H7 strain EDL933 was investigated. Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity. By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E. coli K-12 strains C600 and DH5 _α became positive for hemolysin production. By transformation of recombinant plasmids carrying a 11.9 kb Bam HI fragment and a 5.3 kb Sal I fragment of pSK3 hemolytic activity is revealed when tranformed in E. coli C600 or DH5α DNA-hybridization of pO157 and subclones with the α-hemolysin specific DNA probe was only found under conditions of low stringency. No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes. Our results indicate that a hitherto not described hemolysin belonging to the α-hemolysin family is encoded by the 90 kb plasmid of E. coli O157 strains.     

Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors

We report the development of simple fluorogenic probes that report on the activity of both bacterial and mammalian uracil–DNA glycosylase (UDG) enzymes. The probes are built from short, modified single-stranded oligonucleotides containing natural and unnatural bases. The combination of multiple fluorescent pyrene and/or quinacridone nucleobases yields fluorescence at 480 and 540 nm (excitation 340 nm), with large Stokes shifts of 140–200 nm, considerably greater than previous probes. They are strongly quenched by uracil bases incorporated into the sequence, and they yield light-up signals of up to 40-fold, or ratiometric signals with ratio changes of 82-fold, on enzymatic removal of these quenching uracils. We find that the probes are efficient reporters of bacterial UDG, human UNG2, and human SMUG1 enzymes in vitro, yielding complete signals in minutes. Further experiments establish that a probe can be used to image UDG activity by laser confocal microscopy in bacterial cells and in a human cell line, and that signals from a probe signalling UDG activity in human cells can be quantified by flow cytometry. Such probes may prove generally useful both in basic studies of these enzymes and in biomedical applications as well.     

Specialized functional domains in hemoglobin: dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide

The fluorescence characteristics of 8-anilino-naphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at beta-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the beta-heme pockets was totally quenched by FIA at beta-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the alpha-heme pockets and FIA at beta-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 A for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the alpha 1 chains and the SH group of the beta 1 chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a blue copper protein from Hyphomicrobium denitrificans and its functions in the periplasm

It has been known that the methylotrophic denitrifying bacteria have the specific electron transfer chains, involving in 'methanol oxidation' and 'denitrification', in the periplasm. Recently, a unique blue copper protein (HdBCP) has been isolated from the methanol-grown methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans. HdBCP is a 14.5 kDa protein and contains one copper atom in the molecule. The electronic absorption spectrum of HdBCP exhibits two absorption maxima near 450 and 750 nm comparable with the intense 600 nm band (epsilon(450)/epsilon(600) = ca. 0.9). The rhombic electron paramagnetic resonance spectrum shows clearly that the copper centre is a 'perturbed' type 1 copper geometry. Stopped-flow kinetics indicates that HdBCP accepts efficiently an electron from cytochrome c(L) (k(2) = 4.0 x 10(6) M(-1) s(-1) at 25.0 degrees C), which is a physiological electron acceptor for methanol dehydrogenase. According to cloning and DNA sequencing of the structural gene, the deduced amino acid sequence shows significant similarities with pseudoazurins, which are a physiological electron donor for Cu-containing nitrite reductase from the denitrifying bacteria. Based on these results, we discuss the role of HdBCP in the electron-flow system, which link 'methanol oxidation' and 'denitrification' together.     

Cellular autofluorescence--is it due to flavins?

For many cell types, cellular autofluorescence in the 500-600 nm spectral region can result in a significant background signal for measurements of weakly fluorescent probes. Measurements of fluorescence intensity and spectra of a variety of cell types and assays of neuronal homogenates are presented to demonstrate that this autofluorescence is most likely due to endogenous flavoproteins.     

Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2''-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely 'essential' for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.     

Synthesis, structure and spectroscopic properties of two models for the active site of the oxygenated state of cytochrome P450 [corrected

Two dioxygen adducts of thiolato-iron(II) porphyrins, [K(222)][Fe(TPpivP)(SC6HF4)(O2)] 1a and [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] 2 were synthesized by reaction of O2 with five-coordinate, high-spin, cryptated alkali metal thiolato-iron(II) 'picket fence' porphyrinate. They were characterized by visible and infrared spectroscopy: lambda max (log epsilon) = 360 nm (4), 427 nm (4.69), 560 nm (3.69), 610 nm (3.40) for both compounds; v(16O-16O) = 1139 cm-1 in chlorobenzene and fluorobenzene for 1a and 2. Single crystals of composition [K(222)][Fe(TPpivP)(SC6HF4)(O2)].[K(222)](SC6HF4)(C 6H5Cl)(H2O) 1b were obtained by diffusion of pentane/xylene mixtures into chlorobenzene solutions of 1a at -5 degrees C. Single crystals of composition [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] were obtained by slow diffusion of pentane into benzene solutions of 2. Structures of 1b and 2 were studied at 20 degrees C (1b) and -100 degrees C (1b and 2). 1b: space group P2(1)/c (monoclinic), a = 16.806(5) A (1.6806 nm), b = 14.331(4) A (1.4331 nm), c = 52.000(15) A (5.2000 nm), beta = 92.95(2) degrees, V = 12.507 A3 (12.507 nm3), Z = 4, Dcal = 1.28 g.cm-3 (t = 20 degrees C). The final R1 factor was 0.085 for 5238 reflections having I greater than 3 sigma(I). 2: space group P2(1)/c (monoclinic), a = 13.107(3) A (1.3107 nm), b = 27.055(4) A (2.7055 nm), c = 25.029(4) A (2.5029 nm), beta = 96.84(2) degrees, V = 8812 A3 (8.812 nm3), Z = 4, Dcal = 1.18 g.cm-3 (t = -100 degrees C). The final R1 factor was 0.088 for 6587 reflections having I greater than 3 sigma(I). The iron atom is, in both compounds, bonded to the four porphyrinato nitrogens (Np), the sulfur atom of the axial thiolate and one oxygen atom of the axially end-on bonded dioxygen molecule. The average Fe-Np distance found in 1b [1.994(4) A, 0.1994 nm] is not significantly different from that found in 2 [1.993(3) A, 0.1993 nm]. The Fe-S bond length is 2.367(3) A (0.2367 nm) in 1b and 2.365(2) A (0.2365 nm) in 2. The Fe-O1 distances with the oxygen atom of O2 bonded to iron are respectively 1.837(9) A (0.1837 nm) and 1.850(4) A (0.1850 nm). The end-on bonded O2 molecule is disordered in both complexes 1b and 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

Oxidative iodination of rabbit IgG: localization of markers in an Fc-fragment and effects of modification]

A comparative study of rabbit IgG, both native and modified ones, designed to assess the functional activity of these proteins under oxidative iodination conditions has been carried out. Polyclonal IgG, its antigen-specific fraction and Iodogen as an oxidant were used. Polyclonal antibodies directed against the CH2 domain of IgG, protein A targeted at the CH-2-CH3 domain interface and ferritin testing the conformation of the antigen-binding Fv fragment, were applied as conformational probes for assessing the changes in the IgG conformation. By taking advantage of pepsin proteolysis of [125I]-IgG, from 80% to 92% of the label was found to be localized within the CH3 domain, thus implying the domain-selective nature of iodination, when the degree of modification was below 0.1 atom of iodine per IgG molecule. Yet, when the three above-mentioned conformational probes were used, considerable alterations in the conformation of not only the CH2 domain and CH2-CH3 domain interface, but in the Fv domain being a part of the Fab fragment, were observed. By using competitive enzyme immunoassay for the straightforward comparative evaluation of functional properties of "cold" (native) and 125I-modified IgG, the deleterious effect of the oxidant (Iodogen) rather than iodine atom substitution at the phenolic ring of Tyr residues was shown to be the major determinant of alterations in the IgG molecule.