Characteristics of trypsin-like activity in subgingival plaque samples

Previous studies have demonstrated that the hydrolysis of the trypsin substrate N-benzoyl-DL-arginine-2-naphthylamide (BANA), by subgingival plaque obtained from a single site, correlates best with the numbers and proportions of spirochetes in plaque samples and may serve as an indicator of clinical disease. In this investigation, we determined whether the association between BANA hydrolysis and spirochetes could be obtained in pooled subgingival plaque samples. Concomitantly, the characteristics of this reaction in terms of substrate type and concentration, microbial numbers needed to give a positive reaction as assessed by microscopic counts, rapidity of hydrolysis, and the effect of pH and various additives on the plaque BANA hydrolytic activity have been studied in pooled plaque samples from patients who were periodontally healthy or diseased. In addition, it was determined whether BANA hydrolytic activity found in subgingival plaque reflected contributions from saliva and supragingival plaque. Results indicated that the assay can best be performed with 0.67 mmol/L BANA at pH 7.0. EDTA and CaCl2 gave a slight inhibition and DTT a slight enhancement of the BANA reaction by the pooled plaque suspensions. The majority of the reactions (85%) developed their full color after overnight incubation. BANA hydrolysis was not found in saliva and occurred with much greater frequency in subgingival plaque as opposed to supragingival plaque. Analysis of the data indicated that BANA hydrolysis by pooled subgingival plaque samples is a suitable test for the detection of spirochetes when two or three spirochetes per high microscopic field are present in the sample.