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[Intramolecular mobility of human major histocompatibility complex protein. A spin-label study].

Membranes of human splenocytes were hydrolyzed by papain and extracellular portions of class I and class II HLA antigen molecules were isolated by monoclonal antibodies fixed on Sepharose 4B. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine and the values of rotational correlation times (tau) of labeled proteins were found using dependencies of ESR spectra parameters vs viscosity at constant temperature. The tau-values were equal to 8 ns for class I molecules and 14 ns for class II molecules. These values are 2-3 times lower than predicted for a rigid ellipsoid with mol wt. 50 kDa (about 20 ns). This fact suggests the existence of flexibility of HLA molecules which seems to be important for their biological activity. In this respect extracellular portions of HLA antigen molecules resemble flexible Fc fragments (tau = 12 ns) and differ from rigid Fab fragments (tau = 20 ns) of immunoglobulins G. The values of tau of spin-labeled proteins adsorbed from membrane hydrolysates on IgG-column was equal to 6.5 ns. The proteins adsorbed on lentil lectin column (after isolation of HLA proteins) have the tau-values equal to 9 ns.

Electron Spin Resonance Spectroscopy↗
PubMed1980

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets.

Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane. The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 degrees C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 degrees C, although the La3+ effect was larger and occurred at lower concentrations than that of Ca2+. For example, addition of 1 mM La3+ or 4 mM Ca2+ increased the order parameter of I(12,3)-labeled platelets by 4.3 +/- 1.7% or 2.1 +/- 0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1 mM LaCl3 or 4 mM CaCl2 additions similarly decreased the lipid fluidity at 37 degrees C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2-1 mM) caused rapid aggregation of platelets which were suspended in a 50-mM Tris buffer pH 7.4 that did not contain adenosine.

Blood Platelets↗
PubMed1996

Quantitative cardiac perfusion: a noninvasive spin-labeling method that exploits coronary vessel geometry.

PURPOSE: To quantitate myocardial arterial perfusion with a noninvasive magnetic resonance (MR) imaging technique that exploits the geometry of coronary vessel anatomy. MATERIALS AND METHODS: MR imaging was performed with a spin-labeling method in six arrested rabbit hearts at 4.7 T. Selective inversion of magnetization in the short-axis imaging section along with all myocardium apical to that section produces signal enhancement from arterial perfusion. A linescan protocol was used for validation of flow enhancement. Flow was quantitated from two images and validated with spin-echo (SE) imaging. Regional perfusion defects were created by means of coronary artery ligation and delineated with gadolinium-enhanced imaging. RESULTS: Linescan estimates of T1 obtained at physiologic flows agreed with model predictions. Flow-induced signal enhancement measured on SE images also agreed with expected values. Finally, perfusion abnormalities created by means of coronary artery ligation were detected. CONCLUSION: This spin-labeling method provides quantitative estimates of myocardial arterial perfusion in this model and may hold promise for clinical applications.

Animals↗
PubMed1981

Spin label reduction kinetics, a procedure to study the effect of drugs on membrane permeability: the effects of monosodium urate, dimethyl sulfoxide and amphotericin B.

A method is presented to study the effect of drugs on membrane permeability. It is based on the reduction of a spin label trapped in the internal aqueous compartment(s) of membranes by ascorbate ions added to the bulk aqueous phase. The decay of the electron spin resonance signal of the spin label as a function of time gives an indication of the effect of added agents on the permeability of membranes. To demonstrate the technique, the effect on model membranes of egg phosphatidylcholine of the gout-implicated compound monosodium urate, the aprotic solvent dimethyl sulfoxide and the polyene antibiotic amphotericin B were examined. Monosodium urate did not affect the permeability, casting doubt on a proposed mechanism whereby the agent disrupts the membranes via hydrogen bonding. Dimethyl sulfoxide promoted a gradual increase in rate of solute passage across cholesterol-containing model membranes. Amphotericin B had a pronounced effect on the permeability of cholesterol-containing membranes, causing nearly total loss of paramagnetism immediately after addition. Some aspects of the mechanism of action of the drugs are discussed as well as the advantages and disadvantages of the method. The experiments also allow the evaluation of the effect of surface charge and cholesterol on the dimensions of model membranes.

Amphotericin B↗
PubMed1995

Spin-labelling study of biomembranes in spontaneously hypertensive rats: calcium- and calmodulin-dependent regulation.

1. The present study was performed to investigate alterations in membrane characteristics of spontaneously hypertensive rats (SHR) by using an electron paramagnetic resonance (EPR) and spin-labelling methods. 2. Washed erythrocytes from SHR were examined and compared with erythrocytes from age-matched normotensive Wistar-Kyoto (WKY) rats. 3. The values of outer hyperfine splitting (2T' 11) and that of the order parameter (S) obtained from EPR spectra for a spin label agent (5-nitroxide stearate) were significantly higher in the erythrocytes of SHR than in those of WKY rats. 4. When calcium (Ca2+) was loaded to erythrocytes with a Ca2+ ionophore (A 23187), the order parameter (S) of the EPR spectra showed a greater increase in SHR than in WKY rats. Furthermore, the Ca2+ -induced change in the order parameter (S) of SHR was significantly antagonized by pretreatment of the Ca2+ antagonists (verapamil, diltiazem) and a calmodulin antagonist (W-7). 5. The results show that the erythrocyte membranes of SHR tolerated different spin motions from those of normotensive WKY rats in the EPR study, which might be associated with the idea that the membrane fluidity might be lower in SHR. Furthermore, the data suggest that Ca2+ -calmodulin antagonists may ameliorate the Ca2+ -induced changes in membrane functions in hypertension.

Animals↗
PubMed1987

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.

Adenine Nucleotides↗
PubMed1980

Effect of anesthetics on membrane fluidity tested by two different techniques: EPR spin labels and polarization of perylene fluorescence.

We have investigated the effect of n-butanol on the spectroscopic properties of two types of probes, that are usually employed to detect membrane fluidity, in mitochondrial phospholipid vesicles and mitochondrial membranes. Although both a spin label (16-doxylstearate) and the fluorescent probe perylene detect a higher immobilization of the membrane in comparison with phospholipid vesicles, the effect of butanol in the case of the membrane is strikingly different with the two techniques employed, showing increased fluidity in the case of the spin label and decreased fluidity with the fluorescent probe.

Anesthetics↗
PubMed2003

Membrane-anchoring and charge effects in the interaction of myelin basic protein with lipid bilayers studied by site-directed spin labeling.

Myelin basic protein (MBP) maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers and may also play a role in signal transduction. Site-directed spin labeling was done at eight matching sites in each of two recombinant murine MBPs, qC1 (charge +19) and qC8 charge (+13), which, respectively, emulate the native form of the protein (C1) and a post-translationally modified form (C8) that is increased in multiple sclerosis. When interacting with large unilamellar vesicles, most spin-labeled sites in qC8 were more mobile than those in qC1. Depth measurement via continuous wave power saturation indicated that the N-terminal and C-terminal sites in qC1 were located below the plane of the phospholipid headgroups. In qC8, the C-terminal domain dissociated from the membrane, suggesting a means by which the exposure of natural C8 to cytosolic enzymes and ligands might increase in vivo in multiple sclerosis. The importance of two Phe-Phe pairs in MBP to its interactions with lipids was investigated by separately mutating each pair to Ala-Ala. The mobility at F42A/F43A and especially F86A/F87A increased significantly. Depth measurements and helical wheel analysis indicated that the Phe-86/Phe-87 region could form a surface-seeking amphipathic alpha-helix.

Amino Acid Sequence↗
PubMed1996

Membrane structure of protein kinase C and calmodulin binding domain of myristoylated alanine rich C kinase substrate determined by site-directed spin labeling.

Cysteine-substituted peptides based on the membrane, calmodulin, and protein kinase C binding domain of the myristoylated alanine rich C kinase substrate (MARCKS) were synthesized and derivatized with a sulfhydryl reactive proxyl nitroxide. These spin-labeled peptides were used in combination with continuous wave power saturation electron paramagnetic resonance (EPR) spectroscopy to determine the position and structure of the peptide on membranes containing phosphatidylserine. These peptides bind at the membrane interface, with nitroxide side chains in the central and C-terminal regions lying several angstroms below the level of the head group. In contrast, the N-terminus of the peptide is extended out of the membrane interface so that the two N-terminal residues are positioned on the aqueous side of the head group. When bound to the membrane, the N-terminal segment of this peptide is sensitive to the membrane surface charge density. Higher charge densities decrease the amplitude of side chain motions at the N-terminus and bring this end of the peptide closer to the membrane interface. When the location of successive residues along the bilayer normal is compared, no helical trend is seen, and no evidence for aggregation of the peptide is found. The EPR spectra of double spin-labeled peptides also show no evidence for a helical structure. Thus, these basic peptides are in an extended configuration at the membrane interface with hydrophobic side chains oriented inward toward the membrane hydrocarbon.

Alanine↗
PubMed2000

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Crystallography, X-Ray↗
PubMed1996

Effects of Actinomyces amphiphile on the fluidity of endothelial cells: a spin label study.

Actinomyces amphiphile (AcA) is an amphipathic molecule produced by Actinomyces viscosus that exhibits several biological activities. The effect of AcA on the fluidity and permeability of the plasma membrane in human umbilical vein endothelial cells was analyzed by a spin label method with 5- and 16-stearic acid nitroxide labels (SAL). These labels help to visualize the fluidity at the shallow (5-SAL) and deep (16-SAL) portions of the lipid bilayer. Cells were incubated with and without AcA (control) at 37 degrees C for 6 hours, and membrane fluidity was periodically measured. Another spin label, 4-(N, N-dimethyl-N-hexadecyl) ammonium-2, 2, 6, 6-tetramethyl-piperidine-1-oxyliodine (CAT-16), was also used to assess the physical state of the cell surface. The order parameter of 5-SAL was significantly lower in the cells incubated with AcA than in control cells after the six-hour incubation. The motion parameter of 16-SAL was significantly lower in AcA-treated cells than in controls after 4 and 6 hours of incubation. These findings indicated that the AcA increased the fluidity. There were no significant differences between the AcA-treated and control cells incubated for only 2 hours. In addition, there were no differences in CAT-16 measurements between AcA-treated and control cells. The release of endoplasmic lactate dehydrogenase (LDH) into the medium tended to increase in the AcA-treated vs. the control cells. LDH release increased in both a dose- and time-dependent manner, indicating that AcA increased the permeability of plasma membranes. These findings suggest that AcA alters the biophysical properties of the plasma membranes of endothelial cells, affecting membrane function.

Actinomyces viscosus↗
PubMed1979

A spin-label study of the effect of gamma radiation on erythrocyte membrane. Influence of lipid peroxidation on membrane structure.

Gamma-irradiation of bovine erythrocyte membranes (0.1-4 Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C12 but not at the depth of C5. Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes. On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed. Membrane proteins underwent radiation-induced conformational transitions revealed by maleimide spin label which could be also connected with lipid peroxidation.

Animals↗
PubMed1985

Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules.

Conformational changes induced by ligands and pH in lupine ferrileghemoglobin selectively modified at Tyr-E16 by the imidazolide spin label has been studied by the method of electron spin resonance in the pH range 6-13. It is shown that in the alkaline pH region the bound spin label registers a local conformational transition which precedes the alkaline denaturation of the protein. In aquamet, cyanide and nicotinate complexes of ferrileghemoglobin this transition occurs with pK 10.5, in acetate and azide complexes with pK 11. In all these ligand derivatives the transition is induced by alteration in the ionization state of one group (delta nH+ approximately equal to 1), most probably, the epsilon-amino group of Lys-GH3. The latter is linked with the Glu-A14 residue and this bond is essential for maintaining the native conformation of leghemoglobin. The ligand-induced conformational changes in the vicinity of the label are small and consist, most likely, in some alteration of the mutual arrangement of the AE and GH helical complexes. No correlation has been revealed between the spin state of the heme iron and the conformation of leghemoglobin in the region under study.

Chemical Phenomena↗
PubMed1994

A site-directed spin-labeling study of ligand-induced conformational change in the ferric enterobactin receptor, FepA.

The ferric enterobactin receptor, FepA, is a TonB-dependent gated porin that transports the siderophore ferric enterobactin across the outer membrane of gram-negative bacteria. We have created two site-directed mutants of Escherichia coli FepA, in both cases introducing a cysteine residue into the putative ligand-binding domain. The introduced cysteines were then modified with nitroxide spin labels for structural and dynamic studies using electron spin resonance (ESR) spectroscopy. The mutants were fully functional, as indicated by their ability to grow under iron-limiting conditions, their uptake of [59Fe]enterobactin, and their sensitivity to colicin B. Labeling of the mutant FepA receptors proceeded easily upon incubation with sulfhydryl-specific spin labels, e.g. MTSL, (1-oxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl methanethiosulfonate. In contrast, spin labeling of the two native cysteines (Cys486 and Cys493) within wild-type FepA occurred only after treatment with a thiol reducing agent and partial denaturation in urea, suggesting that the native cysteines are disulfide-linked. ESR spectra showed a high degree of motional restriction for all three sites. Continuous wave (CW) saturation studies indicated that one of the mutationally introduced sites (Cys280) was surface-localized as evidenced by its exposure to the aqueous paramagnetic relaxation agent chromium oxalate and its low accessibility to O2. The other (Cys310) apparently occupies a site near the membrane/aqueous interface. The native cysteines occupy a site tightly packed within the protein structure with low accessibility to both CROX and O2. A shift in both conventional and saturation-transfer ESR spectra of MTSL-labeled E280C and E310C (but not MTSL-labeled wild type) FepA was observed upon addition of ferric enterobactin. The ESR spectral shift was dependent on ferric enterobactin concentration and did not occur with siderophores not recognized by FepA. Ferric enterobactin binding did not alter the CW saturation properties of MTSL bound to these sites, but did influence their accessibility to O2. These results provide consistent evidence for a ligand-specific conformational change in the surface peptides of FepA upon the binding of ferric enterobactin.

Bacterial Outer Membrane Proteins↗
PubMed1976

Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid. A spin label study.

Lipophilin, a hydrophobic protein purified from the proteolipid of normal hupid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electreter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component waicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged.

Brain↗
PubMed1995

Denaturant unfolding of the ferric enterobactin receptor and ligand-induced stabilization studied by site-directed spin labeling.

FepA is an integral outer membrane protein that is the specific receptor for the siderophore, ferric enterobactin, and is thus primarily responsible for iron uptake in many Gram-negative bacteria. A site-specific mutant of FepA, containing a single introduced cysteine in the ligand-binding domain, was spin labeled and used to examine the denaturant-induced unfolding of this receptor with guanidine hydrochloride (Gdn-HCl) and urea. Electron spin resonance (ESR) spectra showed conversion of the spin label from a motionally-restricted, immobilized environment to a freely-accessible, rotationally-mobile state upon denaturation. Unfolding was also followed by nondenaturing polyacrylamide gel electrophoresis (PAGE), which is sensitive to loss of the putative transmembrane beta-structure, and displayed a similar concentration dependence. Unfolding occurred over relatively narrow ranges of denaturant concentration, indicating a high degree of cooperativity. Unfolding was fully reversible under the conditions employed. Rapid, spontaneous refolding occurred in the presence of Triton X-100 and did not require exogenous lipids. Refolding could be induced by either dialysis, dilution to low denaturant concentration, or ethanol precipitation. At ambient temperature the free energy of unfolding extrapolated to zero denaturant concentration (delta GU zero) was 6.24 +/- 0.63 kcal/mol. Values of delta GU zero obtained with Gdn-HCl and urea were in good agreement, as were values obtained from linear extrapolation and nonlinear regression fitting to a two-state equilibrium. This is the first report of a quantitative evaluation of the free energy of unfolding for an integral membrane protein.

Bacterial Outer Membrane Proteins↗
PubMed1981

15N- and 2H-substituted maleimide spin labels: improved sensitivity and resolution for biological EPR studies.

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of (15)N for (14)N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [(2)H]MSL and the (15)N-substituted and deuterated derivative [(15)N, (2)H]MSL were compared with those of the parent MSL. The [(15)N, (2)H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (i) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (ii) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing (14)N with (15)N. In the freely tumbling state, the spectra of [(15)N, (2)H]MSL and [(2)H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD(+) oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [(15)N, (2)H]MSL and [(2)H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [(15)N, (2)H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 musec to 1 msec. The EPR spectrum of [(15)N, (2)H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and g tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the (15)N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by (15)N substitution. Use of (15)N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.

Computers↗
PubMed1981

Hapten-induced changes in pig anti-dansyl antibodies revealed by EPR spectra of spin-labelled antibodies.

Pig anti-Dns antibodies were labelled by 2, 2, 6, 6- tetramethyl-N1-oxylpiperidine-4-amino (N-dichlorotriazine) (label SL I) or by 4-amino-2,2,6,6-tetramethyl-piperidine-N1-oxyl (label SL II). SL I presumably reacted with Fab parts, whereas SL II was attached to the carbohydrate moiety of the Fc part of the antibody molecule. The new method of intramolecular mobility estimation based on viscosity-dependent correlation times of the N-O group of spin-labelled proteins was used, making it possible to separate the correlation times of the spin-label relative to the protein carrier (tauR) and of the labelled protein itself (tauM). The altered shape of the EPR spectrum observed upon binding of epsilon-Dns-lysine indicates that the relative fluctuations of the liganded antibody domains within the Fab part are reduced. The character of temperature dependence of the correlation times of the labelled antibody changed upon hapten binding as well, suggesting an altered interaction between antibody subunits. Evidence of hapten-induced changes of both tauR and tauM was obtained at 1 degrees C and 5 degree C using the label SL II bound to the Fc part. The character of the changes can be interpreted in terms of mobilization of the domains and of altered interaction between labelled carbohydrate and the protein moiety. At 5 degree C the addition of 3% of D2O to the antibody labelled by SL II brought about a substantial effect qualitatively similar to that induced by the hapten.

Animals↗