Rotational dynamics of monoclonal anti-dansyl immunoglobulins

Reidler, Jeffry A.; Oi, Vernon T.; Carlsen, William; Vuong, Thuy-Duong; Pecht, Israel; Herzenberg, Leonard A.; Stryer, Lubert

doi:10.1016/0022-2836(82)90258-3

Cited by 51 publications

(32 citation statements)

References 19 publications

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“…All IgG hinge sequences have similar polyproline cores (see Table I). Yguerabide et al (1970) reported that the fluorescence anisotropy curves of Fab fragments of rabbit IgG anti-DNS antibodies exhibit a single decay component with an appropriate mean rotational correlation time for a globular protein of 50 000 Mr. We also observed only single decay components with Fab fragments from monoclonal mouse IgG, anti-DNS antibodies (Reidler et al, 1982). We suggest that in addition to the inter-heavy chain disulfide bridges, polyproline helical structures may act as restricting elements defining the length of the 'upper hinge' and consequently Fab segmental motion.…”

Section: Discussionsupporting

confidence: 63%

“…We previously showed that independently derived mouse IgG1 anti-DNS antibodies with different combining sites generate different emission spectra (Reidler et al, 1982). This hapten is a sensitive indicator of the polarity of its micro-environment.…”

Section: Resultsmentioning

confidence: 98%

“…As a measure of segmental flexibility, the fluorescence emission anisotropy kinetics of the genetically engineered anti-DNS antibodies were determined using a single-photoncounting instrument described previously by Reidler et al (1982). Excitation with picosecond pulses of polarized light preferentially excites a subpopulation of identically aligned molecules.…”

Section: Segmental Flexibilitymentioning

confidence: 99%

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Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

et al. 1988

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We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen‐combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

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Section: Discussionsupporting

confidence: 63%

Section: Resultsmentioning

confidence: 98%

Section: Segmental Flexibilitymentioning

confidence: 99%

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Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

et al. 1988

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“…Segmental flexibility of IgG molecules has been reported by a variety of methods, including nanosecond fluorescence depolarization assays, [31][32][33] immunoelectron microscopy, 16,20,[34][35][36] X-ray crystallography, 26 nuclear magnetic resonance spectroscopy, 17 small angle X-ray scattering and sedimentation studies 37 and quasi-elastic light scattering. 38 These methods are useful for comparing different molecules, but are inadequate for quantifying the many different modes of flexibility in the molecule.…”

Section: Discussionmentioning

confidence: 99%

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

Correia

Sung

Burton

et al. 2013

mAbs

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“…Originally, ''local'' motion was attributed to mobility of a probe in excess of that expected by the rotation of a rigid body to which it is attached, due to probe motion around its point of attachment to the macromolecule (Wahl & Weber, 1967). Eventually, however, improved methodologies and a better understanding of the dynamic nature of proteins led to the appreciation that ''local'' motion could include internal or domain motions of the protein as well, such as the segmental flexibility attributed to antibodies (Reidler et al, 1982: Oi et al, 1984Hamman et al, 1996). Hones et al (1986), using NADH as the bound fluorophore, reported a rotational correlation time (4) of 27 ns for mMDH at 25 "C, which corresponds to a Debye rotational relaxation time of 81 ns ( p = 34; Jameson & Sawyer, 1995).…”

Section: Discussionmentioning

confidence: 99%

Aggregation states of mitochondrial malate dehydrogenase

et al. 1998

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The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 k 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDHfluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.Keywords: dissociation; fluorescence polarization; mitochondrial malate dehydrogenase: time resolved Porcine heart mitochondrial malate dehydrogenase (mMDH) [(S)-malate:NAD+ oxidoreductase, E.C. 1 .I. 1.371 is a dimeric enzyme composed of identical subunits, each with 314 amino acids, which catalyzes the interconversion of L-malate and oxaloacetate utilizing NAD+ as a coenzyme. In 1970s and 1980s, several groups studied the dimer-monomer equilibrium of mMDH using different approaches. Shore and Chakrabarti (1976) reported fluorescence polarization studies on enzyme labeled with either FITC or fluorescamine and, in both cases, reported finding a concentrationdependent dissociation with a dissociation constant (&) equal to 2 X IOu7 M at 23 "C in pH 8.0, 50 mM Tris-acetate buffer. Bleile et al. (1977) and Hodges et al. (1977) reported gel filtration chromatography and sedimentation velocity ultracentrifugation studies indicating dissociation constants for the dimer similar to those reported by Shore and Chakrabarti. Frieden et al. (1978), however, reported enzyme kinetic studies suggesting that mMDH did not undergo dissociation even at 10" M. Jaenicke et al. (1979), using gel filtration chromatography, also concluded that mMDH remained a dimer over the concentration range of 1.67 X IO-' M to 2.9 X M in 0.2 M phosphate buffer, pH 7.6, 20°C. Wood et al. (1978Wood et al. ( , 1981 and Hodges et al. (1977) reported a pH dependence of the dimer/monomer equilibrium, suggesting that pH values below 7 promote dissociation; specifically, a dissociation constant greater than 2 X lop4 M was reported at pH 5.0, while at pH 7.5 this value was given as less than M (...

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Rotational dynamics of monoclonal anti-dansyl immunoglobulins

Cited by 51 publications

References 19 publications

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

Aggregation states of mitochondrial malate dehydrogenase

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