Identification of a zinc-binding cystic fibrosis antigen in human saliva by 65Zn probing and N-terminal sequencing

Human whole saliva was collected at 4°C into a protein inhibitor cocktail to prevent proteolytic degradation. Saliva was clarified by centrifugation and fractionated by immobilized metal affinity chromatography (IMAC) after charging the column with zinc ions. Proteins with an affinity for zinc were eluted from the IMAC column, characterized by sodium dodecylsulphate-polyacrylamide gel electrophoresis, and probed with 65ZnCl2 after electrotransfer on to polyvinylidenefluoride membranes. A zinc-binding protein of approx. 11 kDa was characterized by N-terminal sequencing followed by a FASTA search of Genbank. The first 40 residues were sequenced, of which the first 34 residues were used to conduct a FASTA search and yielded an homology >97% coding for sequences of mRNA of two proteins, a cystic fibrosis antigen with an Mr of 10,938 and a calcium-binding inflammatory protein MRP8 with an Mr of 10,835 expressed in chronic inflammation. The identity of the last 15 residues of the sequence and the likelihood that the protein is secreted via saliva indicates that the 11-kDa protein is the cystic fibrosis antigen, a protein not previously reported in saliva.