Identification of Carbonic Anhydrase VI with Gustin and Establishment of a Method for Quantitative Measurement of Carbonic Anhydrase VI in Saliva

Objectives: A zinc metallo-protein with molecular weight of 37,000 was isolated from human saliva and called "gustin" due to its possible function in taste. Thereafter, the possibility of Carbonic anhydrase (CA) ‡Y being identical with gustin was reported. This investigation was conducted to reconfirm the identification of CA‡Y with gustin, and to establish a method to measure the concentration of CA‡Y in saliva. Methods: Synthetic peptide designed from 93-111 chain of human CA‡Y was used as a hapten and it was conjugated with KLH to make an antigen. The antibody in the serum was raised in two New Zealand white rabbits by injecting the antigen. Whole saliva, parotid saliva and submandibular-sublingual saliva were collected from 30 healthy individuals respectively. The salivary proteins were separated by two-dimensional electrophoresis, then the proteins were transferred to PVDF membrane and its strip was immunoblotted with the antibody using avidin-biotin complex (ABC) method. Furthermore, the concentration of CA‡Y in saliva was quantified by the enzyme-linked immunosorbent assay (ELISA) using the antibody against the synthetic peptide. ELISA plates were coated by saliva diluted in coating buffer (50mM carbonate), followed by the ABC method. Results: Only one well-defined band around M.W. 37,000 was identified among the proteins from whole saliva samples and parotid saliva ones by the immuno-blotting, whereas it was not recognized in submandibular-sublingual saliva samples. By the ELISA, CA‡Y titers were calculated by reference to the standard curve of the synthetic peptide. CA‡Y in saliva was found among all subjects. Conclusions: These results confirm that gustin is identical with CA‡Y in human parotid saliva, and they suggests that the ELISA using this antibody can be a probe for the quantitative measurement of CA‡Y, which may be useful to diagnose taste dysfunction.