No abstract

No abstract

We have studied the shape of rabbit Immunoglobulin G molecules in solution by using singletsinglet energy transfer to determine the minimum distance between the two hapten binding sites. A hybrid antibody was prepared in which one site specifically bound the energy donor, e-dansyl-lysine, and Although the conformation of IgG molecules has been studied by several techniques, the spatial relationship of the Fab and Fc fragments in solution has not been defined. The results of electron microscopy, transient electric birefringence, and x-ray scattering studies have suggested that the IgG molecule is probably Y-or T-shaped (1-5) with the binding sites located at the ends of the Fab fragments (2, 5). However, it is not known whether there is a preferred angle between the Fab fragments in the free immunoglobulin in solution. If such a preferred angle exists it probably represents an average position, since some freedom of oscillation of the Fab arms about the hinge region has been shown to occur (6). The demonstration of a preferred conformation would help to answer the critical but as yet unresolved question of whether or not a definite conformational change occurs when an antibody binds to a multivalent antigen, and if so, in what direction might such a conformational change lie.We report in this paper the results of our attempt to resolve the matter of a preferred conformation of IgG in solution. We have approached the problem by using singlet-singlet energy transfer to measure the distance between the two hapten binding sites (7). This method has been shown to be t To whom correspondence should be addressed. both theoretically and experimentally applicable to the measurement of molecular dimensions and has been successfully used in investigation of the properties of several macromolecules (7-10).We have prepared a hybrid antibody molecule (11-13) in which one active site specifically bound e-dimethylaminonaphthalenesulfonyl (DNS)-lysine, the energy donor, and the other site bound fluorescein, the energy acceptor. Fig. 1 shows a schematic representation of this molecule. The spectroscopic properties of these antibody-bound haptens are particularly well suited for the observation of long-range resonance-energy transfer between them. The quantum yield of DNS-lysine is considerably enhanced, while that of fluorescein is substantially reduced, resulting in a separation of lifetimes of greater than a factor of ten. Fig. 2 demonstrates that there is highly favorable overlap of the donor emission and acceptor absorption, which, together with the high quantum yield of the donor, makes energy transfer probable over large distances, i.e., in the order of 5-8 nm (5-80 A). MATERIALS AND METHODSHigh affinity antifluorescein and anti-DNS antibodies (15) were elicited by repeated immunizations in the hind toepads of rabbits with fluorescein-hemocyanin (fluorescein isothiocyanate, Isomer I, Sylvana Co, Milburn, N. J., and keyhole limpet hemocyanin, Sigma) or DNS-hemocyanin (DNS-Cl, Pierce Chem. Co.) in complete Freund's a...

Kinetic studies of the interaction of ribonuclease and cytidine 3'-phosphate have been carried out using the temperature-jump method. Three distinct relaxation processes have been observed which are related to the binding of cytidine 3'-phosphate to ribonuclease. The characteristic relaxation times of these processes have been studied as a function of p H , temperature, and concentrations. A quantitative correlation of all of the available data concerning this interaction can be made i f the pH-dependent isomerization of ribonuclease is taken into account. One of the relaxation processes can be associated with the initial complex formation between cytidine 3'-phosphate and ribonuclease. The secondorder rate constant f o r the association reaction is very large ( k N 5 X 108-5 X IO9 M-' sec.-l) and the initial binding process is associated with an unusually large and positive standard entropy change. The p H dependence of the kinetic constants has been interpreted in terms of three ionizable groups at the active site of the free enzyme with p K values of approximately 5 , 6 , and 6.7 at 25". These groups are probably a carboxyl and two imidazole side chains, respectively. The other two relaxation processes are associated with isomerizations of the enzyme-cytidine 3'-phosphate complex. One of these relaxation processes is also observed with ribonucleasecytidine 2'-phosphate and ribonuclease-pyrophosphate complexes; accordingly, it is probably an isomerization involving a catalytically inactive f o r m of the enzymeproduct complex. The large deuterium isotope effect observed on the rate of this isomerization of the ribonuclease-cytidine 3'-phosphate complex at both 25 and 12.5" indicates that a proton transfer is probably involved. One of the relaxation effects is observed only with the ribonuclease-cytidine 3'-phosphate complex and is probably associated with the step prior to the ratedetermining step f o r the formation of cytidine 2',3'-cyclic phosphate f r o m cytidine 3'-phosphate. The results are interpreted in terms of elementary mechanistic steps and a speculative mechanism which accounts f o r the hydrolysis of R N A as well as of cyclic phosphates is presented. IntroductionThe first paper in this series3 presented the results of a kinetic investigation of the interaction of ribonuclease and cytidine 3'-phosphate utilizing the temperature-jump method. Two different relaxation processes were observed: one process was associated with the formation of an enzyme-cytidine 3 '-phosphate complex, while the

No abstract