Affinity labelling of isoelectrofocused fractions from a DNP antibody preparation with the photoactive labels 2,4-dinitrophenyl-1-azide and 2,4-dinitrophenyl-epsilon-aminocaproyldiazoketone.

Isoelectric (I.E.F.) fractions from two DNP antibody preparations have been affinity labelled with the photo-active labels 2,4-dinitrophenyl-1-azide (DNP—N) and 2,4-dinitrophenyl-ε-aminocaproyl diazoketone (DNP—EACDK). The ratios of heavy to light chains labelled for I.E.F. fractions A, B and C of antibody preparation A-2, labelled with DNP-N, were 4.5, 3.1 and 2.4 respectively. In excellent agreement with these results were the ratios found for the labelling of I.E.F. fractions A, B and C of a second antibody preparation A-1 pool I, which were 4.4, 3.8 and 2.0 respectively. A second fraction of DNP antibody preparation A-1 (pool II) was isoelectrically focused, and the I.E.F. fractions A′, B′ and C′ were affinity labelled with the label DNP—EACDK. The ratios of heavy to light chains labelled for the I.E.F. fractions A′, B′ and C′ were 3.4, 0.12 and 5.6 respectively. Mass spectral analyses of the predominant radioactive fraction isolated from the nagarse digestion of the DNP-N labelled heavy chains from I.E.F. fractions A-2 A and A-2 C indicated that fraction A was labelled primarily on a phenyl-alanine residue and that fraction C contained most of the label on an alanine residue. The labelling results support the view that the combining sites of anti-DNP antibodies are distinct and differ substantially and the `characteristic' heavy/light chain ratio of 2 to 4 represents an average of individually homogeneous sites which are labelled by a given affinity reagent exclusively on either light or heavy chains but not both.