| Abstrakt: |
While many workers in recent years have demonstrated the presence of at least three vitamin B12binding proteins in normal serum, human saliva has been described to consist of only one binding protein, namely, transcobalamin I, or the higher molecular weight protein. TC-II and TC-III have not been reported in saliva.In our attempts to standardize a technique of isolating these vitamin B12binders on a small DEAE-cellulose column (1), we also used saliva from patients to determine the effect of different buffers on the pattern of elution of salivary binding proteins. Data, so accumulated, indicate that saliva not only contains both TC-II and TC-III in small amounts, but that the TC-II of saliva is also capable of mediating B12uptake by human lymphocytes transformed with phytohemagglutinin (PHA) in vitro.Materials and methods. Saliva from normal human volunteers, both male and female, were obtained, centrifuged to remove any particulate matter and the clear saliva stored at −20°. Once thawed, samples were not re-used. Radioactive cyanocobalamin ([57Co]B12, sp act 182 mCi/mg) was obtained from Amersham/Searle.DEAE-cellulose column chromatography.DEAE-cellulose was prepared according to Gizis and Meyer (7). To 0.5 ml of saliva, 250 pg of [57Co]B12were added and the mixture was incubated at 37° for 20 min. After counting, the mixture was dialyzed against a liter of 0.0175 Msodium phosphate buffer, pH 6.3, overnight, and subjected to chromatography on DEAE-cellulose columns as described by Kumar et al. (1). The isolated fractions were collected, dialyzed against large volumes of distilled water in the cold for 24 hr and concentrated on a minicon B 15 micro-concentrator (AMICON).Gel-filtration on Sephadex G-200 column.Two milliliters of saliva were incubated with 1000 pg of [57Co]B12at 37° for 20 min and the mixture dialyzed against 4 liters of 0.005 Msodium phosphate buffer, pH 7.4, containing 1 MNaCl, overnight at 4°. |
