Hydrogen-ion-titration curve of rabbit immunoglobulin against ovalbumin.

Abstract

The hydrogen ion equilibria of rabbit immunoglobulin raised against purified ovalbumin, was studied in order to see how its titration behaviour compares with that of non‐specific normal immunoglobulin. Potentiometric as well as spectrophotometric titration of the antiovalbumin was performed at three temperatures, i.e. 15, 25 and 35 °C and at an ionic strength of 0.15. From the nature of the titration curve, it was possible to count and characterise the ionizable groups in the antibody. Thus, the number of different dissociable groups per 160000g antiovalbumin were: 128 carboxyl groups (pKint. = 4.62), 16 imidazole groups (pKint. = 6.3), one α‐amino group (pKint. = 7.5, 71 ɛ‐amino groups (pKint. = 9.72), 56 phenoxyl groups (pKint. = 9.75), and 45 guanidinium groups (pKint. = 11.6) where pKint is the intrinsic pK. Using these values, the titration curve could be generated farely well up to pH 10. The ionization behaviour of carboxyl as well as imidazole groups in the specific immunoglobulin was found to be the same as that for similar groups in normal immunoglobulin. Unlike phenoxyl groups in normal immunoglobulin, 39 phenoxyl groups in antiovalbumin titrated with normal P pKint. and the remaining 17 groups became available for titration only upon alkali‐induced unfolding of the protein molecule at pH 13. The abnormally low value of the electro‐static interaction factor could be attributed to the absence of electrostatic interactions in the molecule and to the possible inadequacy in the theoretical treatment of hydrogen ion equilibria of proteins like immunoglobulin.