Ionization and Reactivities of the Thiol Groups Which Participate in the Formation of Interchain Disulfide Bonds of Bence Jones Proteins and an Fab(t) Fragment1

Abstract

The pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab(t) fragment of a myeloma protein (Jo) (IgGl, kappa) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type kappa Bence Jones protein dimers had the same pK values (pK = 9.76 at 0.2 ionic strength and 25 degrees C) and the same true second-order rate constants (k) toward chloroacetamide (k = 18.8 x 10(-2) M-1 . S-1). The two thiol groups of partially reduced type lambda Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1 = 8.5-8.6 and pK2 = 9.5-9.7 at 0.2 ionic strength and 25 degrees C). The spectrophotometric titration of partially reduced Nag protein (type lambda) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab(t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25 degrees C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type kappa) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, k1 and k2, of the two thiol groups of type lambda Bence Jones proteins varied with the specimen (k1 = 1.9-5.7 x 10(-2) M-1 . S-1 and k2 = 18.5-25.0 x 10(-2) M-1 . S-1). The k1 and k2 values for Jo-Fab(t) were 7.21 x 10(-2) and 23.1 x 10(-2) M-1 . S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.