While a group of oral commensals have been implicated in the aetiology of 
chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas 
gingivalis is most commonly reported to be associated with severe forms of the 
disease. Although a variety of human tissues can produce a number of 
peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine 
residues to citrulline, P. gingivalis is one of the few prokaryotes known to 
express PAD. Protein and peptide citrullination are important in the development 
of rheumatoid arthritis and in recent years a number of authors have suggested a 
possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some 
have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the 
prime purpose of this study was to further characterise PAD in P. gingivalis 
cells particular emphasis on substrate specificity, using arginine containing 
peptides and RA relevant proteins.
METHODS: P. ginigvalis W50 was anaerobically cultured in BHI broth, cells 
harvested and resuspended in assay buffer. A colourimetric assay was developed 
to measure citrulline and employed to determine enzyme activity using the 
substrate BAEE. The assay was employed to investigate the effects of 
environmental pH and temperature on activity. Citrullination of BAEE by 
sonicated cells allowed the proportion of intracellular enzyme to be estimated. 
Enzyme specificity and substrate preference were investigated by using various 
arginine containing peptides, proteins and arginine analogues, as substrates and 
measuring the rate of citrullination. The influence of gingipains on 
citrullination was assessed by measuring the rates of citrullination of bovine 
serum albumin in the presence of protease inhibitors.
RESULTS: Enzyme activity decreased by 13% following exposure of cells to 60 °C 
for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD 
activity is cell surface associated and the remainder cytoplasmic. Optimal pH 
for enzyme activity was between pH 7.5 and 8. All small arginine-containing 
peptides were citrullinated with reaction rates faster than that for free 
arginine with rates that varied with arginine residue position and number. 
Arginine analogues exhibited minimal effect and influence when tested as either 
substrates or competitive inhibitors. Cells were able to citrullinate yeast 
enolase, human vimentin and fibrin at varying rates. All proteins were modified 
at slower rates than those for peptide substrates. Inhibition of gingipains had 
no influence on the rate of protein citrullination.
CONCLUSIONS: P. gingivalis PAD is a primarily cell surface associated, heat 
stable, enzyme that exhibits optimal activity under alkaline conditions similar 
to those present in the inflammatory environment. The enzyme displays high 
specificity for arginine residues in peptides and modified arginine in all 
positions and the gingipains did not influence the rate of protein 
citrullination. The ability of the enzyme to convert arginine residues in all 
proteins tested would indicate that its presence in inflamed tissue may promote 
autoimmune reactions by creation of altered host epitopes.