Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5

Summary The FLAG^® peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identified tag sequences are those that bind only one of the anti-FLAG mAbs and those that bind only two or all three of the anti-FLAG mAbs. Examples of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs M1 and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variants was increased, e.g. using mAb M2 it was found that replacing DDDDK in MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fold. The activity of this peptide was studied in more detail. In different direct ELISAs, in which MDYKAFDNL was synthesized on polyethylene pins, coated onto polystyrene microtiter plates or onto nitrocellulose paper, the activity of this peptide was similar, i.e. increased at least 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA the binding activity of soluble MDYKAFDNL was decreased 10-fold over those of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest that, in solution, the conformation of MDYKAFDNL is more unstructured compared to its conformation when coated or linked to a carrier. We postulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed proteins and/or subunits.Abbreviations ABTS 2,2-azino-di-3-ethylbenzthiazoline sulfonate - ELISA enzyme-linked immunoabsorbent assay - GDA glutardialdehyde - HPLC high-performance liquid chromatography - mAb monoclonal antibody - rampo rabbit-anti-mouse peroxidase - SDS sodium dodecylsulfate     

Specific Interaction of Coproporphyrin I with Antibodies in Water and Reverse Micelles: Dimerization of Antibodies Affects Antigen Binding Site

The antigen-antibody interaction between coproporphyrin I and anti-coproporphyrin antibodies was studied by a fluorescence method in water and a reverse micellar system: n-octane/Aerosol OT. Coproporphyrin fluorescence was quenched, and coproporphyrin emission maximum was shifted to the long-wavelength region after binding to the antibodies or Fab-fragments. The mechanism of this quenching is static, most probably, by a tryptophan residue (or maybe lysine or methionine). Apparent dynamic quenching, in this case, arises from protein backbone motion. A special kind of antibody Fab-Fab dimerization was proposed.     

Fluorescence spectroscopy of monoclonal antibodies produced against the fluorescyl hapten conjugated through the xanthene ring

Two mouse anti-fluorescyl monoclonal antibodies (mAb), clones FL43.1 and FL55.3, were produced to the fluorescein hapten, which was conjugated to the carrier protein through the 4 position of the xanthene ring. Association constants (K _A) and thermodynamic parameters for both mAb were ascertained by monitoring the steady-state intrinsic and fluorescein fluorescence. Both techniques were in good agreement and gaveK _A values in the 10⁹ M ^–1 range. Ligand-induced intrinsic fluorescence quenching showed a hypsochromic shift for mAb FL43.1, but not for FL55.3, suggesting that the ligand interacts with different tryptophan residues in each mAb. Because these mAb are directed toward the phenylcarboxylate portion of fluorescein, the different ionic and structural forms should be useful as indicators of antibody binding site pH and buffering capacity near the binding site.     

Effect of point defect concentration in NaCl and LiF crystals on the saturation field of the magnetoplastic effect

The effect is studied of the calcium impurity concentration in NaCl crystals and of preliminary x-ray irradiation of NaCl and LiF crystals on the magnetic saturation field B₀ characterizing the transition from the conventional proportionality of the dislocation mean path length l to the magnetic induction B squared(l∝B²) to saturation (l=const). B₀ is shown to increase with the calcium concentration in NaCl crystals and with the dose of x-ray irradiation of NaCl and LiF. This finding indicates that the dislocation breakaway from local defects in weak magnetic fields is controlled by the mechanism of longitudinal spin relaxation in a system of radical pairs that form due to interaction between dislocation cores and paramagnetic centers.     

Seventh and ninth order nonlinear susceptibility measurement in alkali metal vapour

We report the first experimental measurement and theoretical estimation of seventh _eff ⁽⁷⁾ and ninth _eff ⁽⁹⁾ order effective nonlinear susceptibilities. The correlation of ninth, seventh and fifth harmonic signal efficiencies in various phase-matched mixtures of Ar (or Xe) and sodium atoms, is experimentally investigated. Using direct and cascade processes of harmonic generation, the modulus ratio is measured for the higher nonlinearities, which allows an estimation of _eff ⁽⁷⁾ and _eff ⁽⁹⁾ from the lower order optical susceptibilities. Results from numerical calculations of atomic nonlinear susceptibilities up to the ninth order, accounting for the discrete spectrum and the continuum, are presented.     

A theoretical investigation of the effect of water on the structure and fluorescence spectra of L-tryptophan

Fluorescence spectra of two long-wavelength electron transitions S₀ ← ¹L_b and S₀ ← ¹L_a in uncharged and zwitterionic forms of L-tryptophan (Trp) in aqueous solution and in the complex of Trp with water molecule were calculated using the Frank–Condon approximation. Geometric parameters of Trp in electronically excited states were determined, and the vibrational structure of vibronic spectra was analyzed. It was shown that the relative position of structural fragments of alanine (R-Ala) and indole (R-In) could have a determining effect on the fluorescence and formation of the vibrational structure of electronic spectra. The increase of the rotation angle between the R-Ala and R-In, which depends on the Trp environment, results in the Trp fluorescence originating only from the singlet excited state ¹L_a.     

A study of the luminescence of glasses activated with holmium

The absorption and luminescence of glasses activated with holmium were studied at room temperature and at low temperatures in the region of 4300–30 000 cm^–1. Using the position of the absorption and luminescence bands, the energy levels and the transition scheme were plotted for the trivalent holmium ion in glass. The influence of the glass composition, of the activator concentration, and of the temperature upon the shape, position, intensity, and lifetime of the 5000 cm^–1 band (⁵I₇⁵I₈) were studied. From the temperature dependence of this band, the scheme for the splitting of the (⁵I₇ and (⁵I₈ holmium levels in glass was obtained.In conclusion, we express our thanks to P. P. Feofilov for this interest.     

On the phase boundaries of the integer quantum hall effect

It has been pointed out that, according to the two-parameter scaling theory, the magnetic-field position of the phases of the integer quantum Hall effect (IQHE) at ω_cτ ? 1 is not determined by the filling factor ν = nh/eB. The position of the IQHE phases is given by the bare Hall conductivity σ _xy ⁰ . In this regard, it has been shown that the diagonal resistivity in the magnetic field measured by Sakr et al. [Phys. Rev. B 64, 161308 (2001)] does not exhibit transitions between the σ _xy = 3, 4 and 6 IQHE states on the one hand and the dielectric state on the other hand in contrast to the assertion by Sakr et al.     

Semiempirical QSAR study and ligand receptor interaction of estrogens

Softness values E_n of estrogen derivatives and softness values E_m of receptor lysine, histidine, tyrosine and cysteine have been evaluated by Klopman equation. The required parameters for the solution of Klopman equation have been calculated with the help of PM3 method. The difference E_nm between E_n and E_m has been derived for QSAR study. The estrogen derivatives have been divided into four different sets on the basis of their structural similarities, and their biological activity taken from literature in terms of relative binding affinity (RBA). The QSAR study shows that, E_nm values provide good relationship with biological activity.     

On the effect of non-parabolicity in the quantum-mechanical treatment of cyclotron resonance in n-Ge

The quantum-mechanical expression for _± including non-parabolicity of the conduction band is applied to the case of the transverse-mass resonance of n-Ge in the quantum regime aroundk _B T~ (=5.6×10¹²s^–1). The essential results concern the resonance position in the transmittivity (shift with temperature) and the line shape (asymmetric absorption).     

Effect of tetrachloroethane on the equilibrium between ionic and molecular complexes in the methylsulfonic acid-butyl acetate system

In concentrated solutions of methylsulfonic acid in butyl acetate (BA), molecular and ionic complexes were found to be in equilibrium:

$AH \cdots B \cdots HA + HA \leftrightarrow AH \cdots BH^ + + (AHA)^ - .$

The base is fully protonated only at a more than 10-fold excess of the acid. In the presence of tetrachloroethane (TCE), a neutral solvent, the IR spectra of the system are indicative of an increase in the concentration of undissociated acid molecules (HA) and a decrease in the concentration of proton solvates (AHA)^?. This means that the equilibrium shifts to the formation of molecular structures. At C _TCE ⁰ :C _BA ⁰ > 2, TCE molecules attenuate the promoting effect of undissociated acid molecules, which are known to facilitate the transfer of a proton to a base molecule with the formation of an (AHA)^? ion with a strong symmetrical H-bond.

     

Spectra of optical absorption and energy levels diagram of Er<Superscript>3+</Superscript> ions in bulk crystals of aluminum nitride

The absorption spectra of the Er³⁺ ions embedded in the AlN matrix have been investigated. The admixture of erbium was introduced in bulk AlN crystals by diffusion. The absorption lines, which are associated with the intraconfigurational electronic f–f-transitions from the ground ⁴ I _15/2-state to the levels of ion Er³⁺ excited states have been observed in the spectral range of 370–700 nm. The transitions to the state levels ⁴ F _9/2, ² H _11/2, ⁴ F _7/2, ⁴ F _5/2, ² H _9/2, and ⁴ G _11/2 have been investigated in detail at the temperature T = 2 K. The number of the observed lines for these transitions coincides with the theoretically possible one for the electronic f–f-transitions in the ions Er³⁺, which are in the crystal field with the symmetry below cubic. The narrowness of the observed lines and their number convincingly testify the replacement of preferably one regular crystalline position by erbium ions. The implementation of Er³⁺ in the Al³⁺ position with the local symmetry C _3v appears the most probable. The energy positions of the levels of excited states for the investigated transitions have been determined. The diagram of the Er³⁺ ion energy levels in the AlN crystals has been built.     

Quantum-Chemical Simulation of the Solvent Effect on Spontaneous Emission of Singlet Oxygen

A molecular simulation of the solvent effect on radiative rate constant k _r of singlet oxygen is carried out. This study included a search for the most probable conformations of the complexes of molecules of singlet oxygen and ten solvents and calculation of dipole moments M of transitions a¹Δ _g –b¹Σ _g ⁺ (M _a–b ) and a¹Δ _g –X³Σ _g ^- (M _a–X ) of the oxygen molecule for them. Averaging of M _a–b by conformations, taking into account the probability of their formation for complexes without atoms with a large atomic number (Cl, S), yields values that, as a rule, correlate well with the behavior of k _r in the experiment. Taking into account the possibility of decreasing the distance (compared to equilibrium) between molecules in a collision complex at room temperature made it possible to achieve satisfactory agreement of the calculated and experimental data also for complexes with CCl₄, C₂Cl₄, and CS₂. The obtained data indicate that a number of factors affect k _r . The correlation of k _r with molecular polarizability in a number of cases is due, on the one hand, to its effect on the strength of dispersion interactions in the complex and, on the other hand, to the fact that it to some extent reflects the position of the upper filled orbitals of the solvent molecule. Both factors affect the degree of mixing of the π orbitals of the singlet oxygen molecule with the orbitals of the solvent molecule, which, as was found earlier, facilitates the activation of the a¹Δ _g –b¹Σ _g ⁺ transition and the borrowing of its intensity by the a¹Δ _g –X³Σ _g ^- transition.     

Surface-enhanced Raman scattering studies on bombesin, its selected fragments and related peptides adsorbed at the silver colloidal surface

SERS studies presented in this work on BN^8-14, [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]BN^6-14, [d-Tyr⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]BN^6-14, BN and its modified analogues, as well as NMB, NMC, and PG-L show that these molecules at pH 8.3 bind to a colloidal silver surface mainly through Trp⁸ and Met¹⁴ residues. Trp⁸ adsorbs at the surface almost perpendicularly. Met¹⁴ appears on the surface mainly as a P_C-G conformer. His¹², as is evident from the spectra, practically does not take part in the adsorption process. Substitution of l-leucine at the 13 position of amino acid sequence with l-phenylalanine does not change substantially the pattern of the adsorption mechanism; however, substitution of phenylalanine at the 12 position (instead of l-histidine) causes changes in the SERS spectra that show that Phe¹² takes parallel orientation to the surface upon adsorption of [d-Phe¹²]BN, while in the case of [Tyr⁴,d-Phe¹²]BN this residue is perpendicular to the surface and influences the orientation of the bound Trp⁸. On the other hand, substitution of Asn with Tyr in the 6 position in nonapeptide fragment causes changes in the adsorption mechanism. In this case, the discussed fragment binds to the silver colloidal surface by Tyr⁶, Trp⁸, and Met¹⁴. The SERS spectrum of NMC is very similar to that of BN; although it differs by the binding orientation of the amide bond towards the surface. Appearance of Phe¹³ in NMB and PG-L causes that this residue competes successfully with Trp⁸ forcing it to take tilted orientation. As seen from the enhancement of the characteristic Phe vibrations this moiety in NMB and PG-L adsorbs on the silver surface in a tilted fashion. This arrangements cause that the 8-14 peptide chain in all these studied compounds takes almost a parallel orientation to the surface while the 1-5 fragment of the peptide chain is removed from the silver surface vicinity.     

The limit of stability of proton-rich thallium isotopes: a search for the decay of177Tl

The fusion reaction⁷⁴Se+¹⁰⁶Cd¹⁸⁰Pb^* at a compound nucleus excitation energy E_x 40 MeV, has been used in a search for the decay of the unknown nucleus¹⁷⁷Tl. Evaporation residues were velocity and mass analysed prior to implantation into a position sensitive silicon surface barrier detector. No evidence was discovered for the proton or alpha-decay of¹⁷⁷Tl for a cross-section > 10 _– ⁵⁺¹⁰ nb. This indicates either that¹⁷⁷Tl decays too rapidly (t_1/2 < 1=">s) by proton emission for the decay to be detected, or that¹⁷⁷Tl is produced with a cross-section less than the limit established in the present experiment. The following nuclear decay half-lives were measured with improved accuracy;¹⁷⁷Hg (t_1/2 = 130±5 ms),¹⁷⁸Hg (t_1/2=250±25 ms) and¹⁷⁷Au (t_1/2=1180±70 ms). The experiment also provided the first direct confirmation of the correct mass assignment of the alpha-decay, E=6.26 MeV, to the decay of¹⁷⁶Au. Two additional halflife measurements of proton-rich Osmium isotopes are also presented from a previous similar experiment;¹⁶⁵Os (t_1/2=73±8 ms) and¹⁶⁶Os (t_1/2 = 194±17 ms).     

Distance distributions and dynamics of a zinc finger peptide from fluorescence resonance energy transfer measurements

Time-resolved fluorescence resonance energy transfer (FRET) measurements were used to measure distance distributions and intramolecular dynamics (site-to-site diffusion) of a 28-residue single-domain zinc finger peptide in the absence and presence of zinc ion. Energy transfer was measured between TRP₁₄ and a N-terminal DNS group. As expected, the TRP-to-DNS distance distribution for zinc-bound peptide is shorter and narrower (R _av=11.2 Å,hw=2.8 Å) than the metal-free peptide (R _av=20.1 Å,hw=14.5 Å). The degree of mutual donor-to-acceptor diffusion (D) was also determined for these distributions. For zinc-bound peptide there is no detectible diffusion (D0.2 Ų/ns), whereas for metal-free peptide a considerable amount of motion is occurring between the donor and the acceptor (D=12 Ų/ns). These results indicate that the zinc-bound peptide folds into a unique, well-defined conformation, whereas the metal-free conformation is flexible and rapidly changing. The absence of detectible mutual site-to-site diffusion between the donor and the acceptor in the metal-bound zinc finger peptide indicates that intramolecular motion is essentially frozen out, on the FRET time scale, as a consequence of zinc coordination.Dedicated to the memory of Barbara D. Wells.     

Evidence for incipient O dimerization and absence of Cu+3 in temperature-dependent XPS of highT c superconducting oxides

From temperature-dependent X-ray photoemission (XPS) spectra between 300 and 10 K of Sr doped La₂CuO₄ and 123 compounds (YBa₂Cu₃O_7–) we deduce the absence of the Cu3d ⁸(Cu³⁺) configuration in the ground state of these Mott insulators. The metallic character of these samples and involving those Cu atoms which are relevant for the superconducting properties arises from (d ⁹ p ⁶)+(d ¹⁰ p ⁵) valence fluctuations, while the superconducting transition is indicated to be closely related to an incipient oxygen dimerization which is best detected upon cooling far belowT _c .     

Processes of associated production of the Higgs boson with the Z boson in a superstrong magnetic field

We compute probabilities for the processes ee+Z+H, and e⁺+e^–Z+H in a superstrong magnetic field. It is shown that the magnetobremsstrahlung of the Higgs boson with the Z boson in the magnetic field Bs>B₀=m²/e=4.41·10¹³ G may be a relatively probable process, and that the superstrong magnetic field significantly influences the process e⁺+e^–Z+H, which is possible even in the absence of the field.Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 1, pp. 104–108, January, 1991.The author thanks V. Ch. Zhukovskii and A. V. Borisov for discussions on the results of this work.     

Laser- and gamma-ray induced crystallization of IR-transmitting calcium gallate glass

Ar⁺-laser (=488 nm) irradiation of calcium gallate (CG) glass with the composition of 60CaO·39Ga₂O₃·Fe₂O₃ resulted in a distinct decrease in the IR transmittance (T) due to the formation of crystalline CaGa₂O₄ and CaGa₄O₉ phases. The Mössbauer spectrum of non-irradiated glass comprised a broad doublet due to distorted Fe³⁺(T_d) with, , and of 0.20, 1.33, and 1.00 mm s^–1, respectively. An additional doublet due to Fe³⁺(T_d) was observed in the Ar⁺-irradiated glass and, , and were 0.17, 1.32, and 0.75 mm s^–1, respectively. A decrease inT was also observed after the⁶⁰Co -ray irradiation with doses 10⁵Gy, and the precipitation of CaO, Ga₂O₃, and CaGa₄O₇ phases was confirmed by X-ray diffraction.     

Influence of the ground state of the Pr<Superscript>3+</Superscript> ion on magnetic and magnetoelectric properties of the PrFe<Subscript>3</Subscript>(BO<Subscript>3</Subscript>)<Subscript>4</Subscript> multiferroic

The magnetic, magnetoelectric, and magnetoelastic properties of a PrFe₃(BO₃)₄ single crystal and the phase transitions induced in this crystal by the magnetic field are studied both experimentally and theoretically. Unlike the previously investigated ferroborates, this material is characterized by a singlet ground state of the rare-earth ion. It is found that, below T _N = 32 K, the magnetic structure of the crystal in the absence of the magnetic field is uniaxial (l ∥ c), while, in a strong magnetic field H ∥ c (H _cr ～ 43 kOe at T = 4.2 K), a Fe³⁺ spin reorientation to the basal plane takes place. The reorientation is accompanied by anomalies in magnetization, magnetostriction, and electric polarization. The threshold field values determined in the temperature interval 2–32 K are used to plot an H-T phase diagram. The contribution of the Pr³⁺ ion ground state to the parameters under study is revealed, and the influence of the praseodymium ion on the magnetic and magnetoelectric properties of praseodymium ferroborate is analyzed.