THE REACTIVITY OF PROTEINS OF SOME CONNECTIVE TISSUES AND EPITHELIAL STRUCTURES WITH 2,4-DINITROFLUOROBENZENE

EUGENIO ZERLOTTI ¹ and MILTON B. ENGEL ¹

¹ University of Illinois, College of Dentistry, Departments of Applied Materia, Medica and Therapeutics and Orthodontics

2,4-Dinitrofluorobenzene was used without any coupling procedure as a histochemical reagent to localize some reactive groups of proteins in connective tissues and epithelial structures. Tissues were fixed by freezing-drying to avoid uncontrolled alterations of proteins and the reactions were modified in some sections by postfixation or pre-treatment with a variety of reagents, including alcohol, formalin, formaldehyde vapors, acetic anhydride, nitrous acid and N-ethylmaleimide. The staining and resolution were greatly enhanced by viewing the sections with monochromatic light at 410 mµ. When used in this way, 2,4-dinitrofluorobenzene is specific for: (1) -amino groups of terminal amino acids; (2)-amino groups of lysine and hydroxylysine; (3) sulfhydryl groups of cysteine. However, the reaction seems to be dominated by the -amino groups of lysine in most of the tissues. In the native state not all functional groups of tissue colloids react presumably because the groups are masked by the interractions involved in forming tertiary structures or covalent bonds. This is specially notable in calcified tissues. Pyknotic nuclei, pre-dentin, carious dentin and newly formed bone are more reactive suggesting a less highly organized state of their structural proteins or some other modification in the lysine and hydroxylysine containing proteins. 2,4-Dinitrofluorobenzene is valuable as a specific cytochemical reagent and, in some instances, as an indicator of the state of organization of cellular and extracellular colloids.

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