A COMPARISON OF CYSTEINE AND SERINE PROTEINASES IN HUMAN GINGIVAL CREVICULAR FLUID WITH TISSUE, SALIVA AND BACTERIAL ENZYMES BY ANALYTICAL ISOELECTRIC-FOCUSING

Gingival crevicular fluid (GCF) contains several different proteinase activities and the study sought to clarify their sources. Gingival tis sue and GCF were collected from chronic periodontitis patients. Gel-fi ltration chromatography of crude tissue extracts yielded cathepsin B a nd tryptase fractions sensitive to cysteine and serine proteinase inhi bitors, respectively. Cell sonicates of suspected periodontal pathogen s were prepared from broth cultures of reference strains. Of these, Po rphyromonas gingivalis showed much the strongest activity and this had an effector response consistent with the metal-dependent cysteine pro teinase described by others. Banding patterns in GCF, tissue and bacte rial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. On Z-Val-Lys-Lys-Arg-AFC overlay s, GCF had bands corresponding to tissue cathepsin B and the enzyme fr om P. gingivalis, though a contribution from Treponema denticola could not be ruled out. Use of D-Val-Leu-Arg-AFC overlays showed GCF activi ty similar to tissue tryptase. In GCF there were additional bands that did not correspond to any tissue or bacterial samples and on Z-Ala-Al a-Lys-AFC overlays these closely resembled activity in parotid saliva. The results confirmed that GCF contains tissue cathepsin B and trypta se, while the apparent presence of enzymes from P. gingivalis and poss ibly T. denticola is consistent with previous reports linking activity to these organisms. The saliva bands demonstrated that contamination of GCF may occur despite rigorous collection procedures. Copyright (C) 1996 Elsevier Science Ltd.