Kozo Hamaguchi

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

Abstract The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism. The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U 1 U 2 N, where U 1 and U 2 are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U 2 to U 1 was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U 1 U 2 reaction is proline isomerization. The rates of interconversion between N and U 2 changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.

Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain: Kinetic role of the intrachain disulfide bond

Abstract The constant fragment of the immunoglobulin light chain whose intrachain disulfide bond is reduced (reduced C L fragment) assumes a conformation very similar to the intact C L fragment (Goto & Hamaguchi, 1979). The kinetics of reversible unfolding and refolding of the reduced C L fragment by guanidine hydrochloride at pH 7.5 and 25 °C were studied and were compared with those of the intact C L fragment described in the accompanying paper (Goto & Hamaguchi, 1982). Tryptophyl fluorescence, far-ultraviolet circular dichroism, and reactivity of the SH groups toward 5,5′-dithiobis-(2-nitrobenzoic acid) were used to follow the kinetics. The results obtained were thoroughly explained on the basis of the three-species mechanism, U 1 U 2 N, where U 1 and U 2 are slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The rate constants of interconversion between U 1 and U 2 and the rate constant for the process from N to U 2 were very similar to the respective values for the intact C L fragment. Only the rate constant for the process from U 2 to N was greatly different between the intact and reduced C L fragments; the rate constant for the reduced C L fragment was about 100 times smaller than that for the intact C L fragment. These results indicated that the slow isomerization of the unfolded molecule is independent of the presence of the disulfide bond and that the kinetic role of the intrachain disulfide bond is to accelerate the folding process. This kinetic role in the folding of the C L fragment was explainable only in terms of the decreased entropy in the unfolded state of the intact C L fragment due to the presence of the disulfide bond.

Formation of the intrachain disulfide bond in the constant fragment of the immunoglobulin light chain

Abstract Regeneration by glutathione of the constant fragment of the immunoglobulin light chain was studied in the absence and presence of 8 m -urea. The species that appeared during the reaction of the reduced constant fragment with oxidized glutathione were trapped by alkylation with iodoacetamide and identified by electrophoresis in 15% polyacrylamide gel at pH 9.5. The kinetics of the reactions starting from various species formed during the reaction of the reduced constant fragment were also studied, and the overall reaction kinetics of the formation of the intrachain disulfide bond in the constant fragment were established in the absence and presence of urea. The reaction of the reduced constant fragment with oxidized glutathione was much slower but the yield of the constant fragment with the disulfide bond was much higher in the absence than in the presence of 8 m -urea. The slowness of the reaction in the absence of urea is due to the two cysteinyl residues of the reduced constant fragment being buried in the interior of the molecule and because oxidized glutathione is capable of reacting with the thiols only in the opened form of the protein molecule. The high yield is due to the cysteinyl thiol and the mixed disulfide in the intermediate forming an intrachain disulfide bond through thiol-disulfide interchange, the reaction sites being exposed to solvent and located at the appropriate proximity. These findings indicate first, that the appropriate proximity of a pair of cysteinyl residues is essential to form a disulfide bond and second, that they are not easily oxidized to disulfide if they are buried in the interior of the protein molecule.

Studies on γ Globulin of Rice Embryo:Part V Secondary Structure of γ1 Globulin

The secondary structure of γ1 globulin from rice embryo was investigated by means of optical rotatory dispersion, circular dichroism and infrared spectroscopy. The optical rotatory dispersion curve of the native γ1 globulin gave a trough at 233mμ with a [m′]233 value of −2,100°, and the Moffitt-Yang plot gave the parameters of a0= −237 and b0= −20. These data suggest the presence of 3% helix and 38%β structure in the molecule. Circular dichroism exhibits a negative extremum at 218 mμ, giving a [θ]R value of −3,730, which suggests the presence of 16°β structure. Infrared spectrum of a thin film of γ1 globulin showed absorption bands at 695 and 660 cm−1 with a small hump at 615 cm−1 characteristic of the β structure, random coil and α helix, respectively. The protein in heavy water exhibits the absorption maximum at 1,630cm−1 which is also characteristic of the β structure.