Isolation and characterization of three non-mucinous human salivary proteins with affinity for hydroxyapatite.
The isolation and chemical analysis of three human salivary proteins is reported. Using cation-exchange, FPLC anion-exchange chromatography and gel filtration, three human salivary proteins with affinity for hydroxyapatite were isolated, having molecular weights of 60 kD, 20 kD and 14 kD, respectively. The 60 kD protein was identified as albumin, and the 14 kD protein as a member of the cystatin family. Isoelectric focusing of the 14 kD protein revealed a single band, having an isoelectric point (pl) of 4.7. The third protein, not described yet, is appearing as a 20 kD doublet on SDS-PAGE. Isoelectric focusing resolved the 20 kD protein into four bands having pl's of 4.8, 5.0, 5.2 and 5.4, respectively. All bands were recognized by monoclonal antibodies to the 20 kD protein indicating that these four protein bands share the same epitope. The 20 kD protein is a glycoprotein with a carbohydrate content of 13% and a molar ratio of Fuc: Man: Gal: GlcNAc: NeuAc = 3.4:2.6:2.9:4.0:0.4. All three proteins bind strongly to a hydroxyapatite-based HPHT column at pH 6.0. With increasing pH, the binding diminished, especially of the 14 kD protein.
Rathman
W M
WM
Department of Oral Biochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Van Zeyl
M J
MJ
Van den Keybus
P A
PA
Bank
R A
RA
Veerman
E C
EC
Nieuw Amerongen
A V
AV
D000418:Albumins / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015891:Cystatins / Q000032:analysis*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006882:Hydroxyapatites / Q000378:metabolism*; D015151:Immunoblotting; D007525:Isoelectric Focusing; D012471:Salivary Proteins and Peptides / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
D000418:Albumins; D000596:Amino Acids; D015891:Cystatins; D006882:Hydroxyapatites; D012471:Salivary Proteins and Peptides; D017886:Durapatite