Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au

Abstract

The dimerization of the variable fragment of the Bence-Jones protein Au was examined in phosphate buffers at pH 6.8 -6.9 and ionic strength of 0.1 M or 0.2 M at 20 "C. The dimerization constant was about 1 x 10' M-'. The reaction enthalpy was positive and the process was entropy driven. The association and dissociation rate constants were 9 x lo6 M-' s-' and 1.5 x lo2 s-l respectively. Temperature-jump experiments exhibited the presence of two isomers of the dimer, which are present at equilibrium in a ratio of about 1 : 1. Isomerization occurred with a half-life of about 0.1 s.Recent X-ray crystallographic studies have shown that immunoglobulin molecules consist of several domains and implied the presence of non-covalent interactions between the homologous domains on different chains [l, 21. These interactions can be studied with isolated fragments. The association between heavy and light chains [3,4] and the dimerization of light chains [5,6] have been studied. The study of the dimerization of the variable fragment will provide information about the interactions between constant domains as well as those between variable domains, when combined with the studies on the whole light chains [5,6]. In the present study both equilibrium and kinetic aspects of dimerization of the variable part of a Bence-Jones protein (Au) were examined. Such data are of interest for an understanding of the interaction between the different domains in a Bence-Jones protein and possibly in immunoglobulin G. It was also hoped that the study may contribute to the important question [1,2] of whether distinct conformational isomers exist for the domains. MATERIALS AND METHODSThe variable part of the Bence-Jones protein Au was prepared as reported [7]. Both the fraction 111 obtained by Sephadex chromatography (G-75, eluent : 0.5 M acetic acid) and the fraction obtained by trypsin digestion were used. Identical results were obtained with both materials. The samples were additionally purified by DEAE-cellulose chromatography (gradient: 0.05-0.4 M Tris-HCI pH = 8.0). These samples showed only one band in dodecylsulfate polyacrylamide electrophoresis. Other chemicals were from Merck and used without further purification.A phosphate buffer solution 0.025 M KH2P04+ 0.025 M Na2HPO4 (pH = 6.9, I = 0.1 M) was used throughout the equilibrium measurements unless otherwise stated. In the kinetic measurements buffer solution of double concentration was used (pH = 6.8, 1 = 0.2 M). The total protein concentration is defined by cp = 2 CD + cM, where L'U (cM) are molar concentrations of dimers (monomers). The concentration of the protein was calculated from the absorbance at 278 nm using a molar absorption coefficient per monomer of 2 . 0~ lo4 M-' cm-'. This value was determined on a weight basis for the solutions of a concentration range of 0.01 -0.02 mM. The effect of dimerization reaction was corrected if necessary. The sedimentation equilibrium measurements were made with a Beckman Spinco Model E analytical ultracentrifuge equipped with a scanner. Rotor ...

Folding of an antibody variable domain in two functional conformations in vitro: calorimetric and spectroscopic study of the anti-ferritin antibody VL domain

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Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

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Effect of Association State and Conformational Stability on the Kinetics of Immunoglobulin Light Chain Amyloid Fibril Formation at Physiological pH

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