Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins.

We have characterized a series of genetically engineered chimeric human IgG3 and IgG4 anti-dansyl (DNS) antibodies with identical antibody-combining sites but different hinge region amino acid compositions to determine how the hinge region influences Fab fragment segmental flexibility, C1q binding, and complement activation. Our data support the correlation between "upper hinge" length and Fab segmental flexibility; moreover, we confirm that a hinge region is essential for C1q binding and complement activation. However, the hinge length by itself is not sufficient for complement activity in IgG molecules. We have demonstrated that the IgG4 hinge, which imparts restricted segmental flexibility, reduces the ability of IgG3 molecules to activate complement. We also find that the IgG3 hinge region, which imparts greater segmental motion, is not sufficient to create complement activation activity in IgG4 anti-DNS antibodies. Finally, we conclude that (i) segmental motion is correlated with "upper hinge" length, (ii) hinge length and segmental flexibility is not enough to alter complement binding and activation, and (iii) segmental flexibility does not correlate with proficiency to activate the complement cascade.

Absence of a bicarbonate-depletion effect in electron transfer between quinones in chromatophores and reaction centers of Rhodobacter sphaeroides.

Higher plants, algae, and cyanobacteria are known to require bicarbonate ions for electron flow from the first stable electron acceptor quinone QA to the second electron acceptor quinone QB, and to the intersystem quinone pool. It has been suggested that in Photosystem II of oxygenic photosynthesis, bicarbonate ion functions to maintain the reaction center in a proper conformation and, perhaps, to provide the protons needed to stabilize the semiquinone (QB-). In this paper, we show that bicarbonate ions do not influence the electron flow, from the quinone QA to QB and beyond, in the photosynthetic bacterium Rhodobacter sphaeroides. No measurable effect of bicarbonate depletion, obtained by competition with formate, was observed on cytochrome b-561 reduction in chromatophores; on the flash-dependent oscillation of semiquinone formation in reaction centers; on electron transfer from QA- to QB; or on either the fast or slow recovery of the oxidized primary donor (P+) which reflects the P+QA- ----PQA or the P+QB- ----PQB reaction. The lack of an observed effect in Rhodobacter sphaeroides in contrast to the effect seen in Photosystem II is suggested to be due to the amino-acid sequence differences between the reaction centers of the two systems.

Interaction of IgE with its high-affinity receptor. Structural basis and requirements for effective cross-linking.

Structural interactions between IgE and its high-affinity receptor have been investigated with the methods of fluorescence resonance energy transfer and genetic engineering. The results indicate that IgE has a bent conformation when bound to receptor on the cell surface and that the site of interaction is contained in the C epsilon 2 and C epsilon 3 domains; the C-terminal domain, C epsilon 4, is not required for binding. Cross-linking of IgE-receptor complexes is required for signal transduction across the plasma membrane. Binding studies with defined bivalent ligands indicate that structural and/or kinetic features determine the functional effectiveness of the cross-linked states.

Charge recombination from the P+QA- state in reaction centers from Rhodopseudomonas viridis.

The rate of decay of the flash-oxidized primary electron donor, P+, from the state P+QA- was studied in reaction centers from Rhodopseudomonas viridis, containing only the primary menaquinone electron acceptor (QA). At 295 K, in 100 mM NaCl and in the presence of o-phenanthroline, the rate of recombination was 470 +/- 15 s-1 at pH 7 and 570 +/- 20 s-1 at pH 9. The rate at ambient temperatures varied somewhat with viscosity, pH and ionic strength. Between 310 K and 275 K, the temperature dependences of the rate, at pH 7 and pH 9, were linear in an Arrhenius plot, with apparent activation energies of 0.20 eV and 0.16 eV, respectively. At lower temperatures, however, the dependences deviated from this behavior. In 60% glycerol (pH 7) the recombination rate was 370 +/- 10 s-1 at 295 K. As the temperature was lowered, the rate decreased but leveled off to a value of 105 +/- 5 s-1 at 170 K and was independent of temperature from 170 K to 100 K. In 60% ethylene glycol, the temperature dependence was similar, but the rate fell to a minimum of 75 s-1 at 170 K and then increased slightly at lower temperatures; it finally became temperature independent, with a value of about 100 s-1, at 110 K. The overall temperature dependence is consistent with charge recombination by two competing pathways: a direct electron-tunneling process which dominates at low temperature (less than 250 K), and a thermally activated process via a higher energy state, M, which decays rapidly to the ground state. The indirect route dominates at high temperature (above 250 K). Taking into account the contribution from the low-temperature pathway, the activation energy (enthalpy) for the activated process, in aqueous buffer, was determined to be 0.25 eV (at pH 7) and 0.19 eV (at pH 9). A likely candidate for M is P+I- (PF), where I is the intermediate bacteriopheophytin electron acceptor, and energetic arguments are presented in favor of this assignment. If a rate of decay of P+I- to the ground state, derived from the experimental value, was used in the description of the thermally activated P+QA- recombination process, the free-energy gap separating M and P+QA- could be estimated to be 0.27-0.28 eV, placing it about 0.95 eV above the ground state and 0.30 eV below the excited singlet state, P*.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis.

The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P(+), a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c 558) and two low potential (Cyt c 553), act as secondary electron donors to P(+). Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c 558 by P(+) with a rate of 3.7×10(6)s(-1) (τ=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×10(5)s(-1) (τ=3.5 µsec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with α-band maxima at 559 nm (Cyt c 559) and 556.5 nm (Cyt c 556), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c 556→Cyt c 559→P(+). At lower redox potentials, a low midpoint potential cytochrome, Cyt c 553, is preferentially oxidized by P(+) with a rate of 7×10(6)s(-1) (τ=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c 559, and it is suggested that a likely arrangement for the four hemes is: c 553 c 556 c 553 c 559P.

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones.

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.

The acceptor quinone complex of Rhodopseudomonas viridis reaction centers.

The acceptor complex of isolated reaction centers from Rhodopseudomonas viridis contains both menaquinone and ubiquinone. In a series of flashes the ubiquinone was observed to undergo binary oscillations in the formation and disappearance of a semiquinone, indicative of secondary acceptor (QB) activity. The oscillating signal, Q-B, was typical of a ubisemiquinone anion with a peak at 450 nm (delta epsilon = 6 mM-1 X cm-1) and a shoulder at 430 nm. Weak electrochromic bandshifts in the infrared were also evident. The spectrum of the reduced primary acceptor (Q-A) exhibited a major peak at 412 nm (delta epsilon = 10 mM-1 X cm-1) consistent with the assignment of menaquinone as QA. The Q-A spectrum also had minor peaks at 385 and 455 nm in the blue region. The same spectrum was recorded after quantitative removal of the secondary acceptor, when only menaquinone was present in the reaction centers. Spectral features in the near-infrared due to Q-A were attributed to electrochromic effects on bacteriochlorophyll (BChl) b and bacteriopheophytin (BPh) b pigments resulting in a distinctive split peak at 810 and 830 nm (delta epsilon = 8 mM-1 X cm-1). The menaquinone was identified as 2-methyl-3-nonylisoprenyl-1,4-naphthoquinone (menaquinone-9). The native QA activity was uniquely provided by this menaquinone and ubiquinone was not involved. QB activity, on the other hand, displayed at least a 40-fold preference for ubiquinone (Q-10) as compared to menaquinone. Thus, both quinone-binding sites display remarkable specificity for their respective quinones. In the absence of donors to P+, charge recombination of the P+Q-A and P+Q-B pairs had half-times of 1.1 +/- 0.2 and 110 +/- 20 ms, respectively, at pH 9.0, indicating an electron-transfer equilibrium constant (Kapp2) of at least 100 for Q-AQB in equilibrium QAQ-B. Also observed was a slow recombination of the cytochrome c-558+ Q-A pair, with t 1/2 = 2 +/- 0.5 s at pH 6.