Conjugation of dinitrofluorobenzene to plasma proteins in vivo in the rat.

The extent of protein dinitrophenylation was determined in plasma and other tissues of anesthetized rats after administration of the model immunogen [3H]dinitrofluorobenzene (DNFB) (25 mg/kg; 5-25 microCi). DNFB was given by the intravenous, intraportal, intramuscular, or oral route. Irreversible binding was determined radiometrically after exhaustive solvent extraction of plasma or organ proteins. The extent of binding was high in plasma after parenteral administration (approximately 1% dose/ml plasma), but less (approximately 0.1% dose/ml) if DNFB was given orally. Low levels of radioactivity were bound irreversibly in liver (0.01-0.13% dose/g) and kidney (0.03-0.10% dose/g) and only residual amounts in other organs. Western blotting was used to identify target proteins in plasma, liver, and kidney using a specific antidinitrophenyl antiserum. No dinitrophenylation could be detected in liver or kidney samples, but strong recognition of two protein bands was observed in plasma. Bands with the same apparent molecular masses (67 and 44 kDa) were seen when DNFB was incubated with rat plasma in vitro. Preliminary evidence for these proteins being albumin and alpha 1-acid glycoprotein, respectively, is presented. The latter may be important for interindividual variability in immune responsiveness, because it is an acute phase protein whose levels fluctuate widely during disease states.