Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9),two calcium-binding proteins of the S100 family

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-we ight proteins found in human gingival crevicular fluid (GCF), but absent fr om serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PACE) analysis. GCF, serum, and whole saliva were coll ected from periodontitis and healthy subjects, as well as from edentulous a nd newborn subjects. Protein samples were separated by two-dimensional poly acrylamide gel electrophoresis, stained with silver, and compared with refe rence protein maps in the SWISS-2D PAGE database. In GCF and saliva from pe riodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in seru m and were less visible in saliva from edentulous and newborn subjects. Fro m N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (S100A8), a member of the S100 family of calcium-binding proteins. Using p eptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other tw o protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The pre sence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclon al antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF repr esent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sul cus and could represent possible markers for periodontal diseases.