Molecular Weight, Optical Rotation, and Tryptic Susceptibility Studies on Modified Fetuins.

The Lul-glycoprotein present in calf fetal serum, first described by Pedersen (1) and termed fetuin, may be isolated by ammonium sulfate fractionation (2) as well as by ethanol precipitation in the presence of Ba++ and Zn+f (3). The amino acid and carbohydrate composition has been determined for salt-prepared fetuin by Fisher, O’Brien, and Puck (4) and for the ethanol material by Spiro and Spiro (5). The percentage composition in each case is very similar, although differences were reported for the sialic acid content. This feature is re-examined in this study, and some additional molecular and structural properties of fetuin are also reported, following partial or essentially complete removal of the sugar acid. A dramatic difference between s&-prepared and ethanolprepared fetuins, from the same serum, was observed in terms of their behavior to proteolytic attack by trypsin and a-chymotrypsin (6). The salt material was resistant to attack, confirming the finding of Fisher, Puck, and Sato, (2), while the ethanol material was rapidly digested, as previously noted by Spiro (7). On the basis of this observation, it was concluded that the ethanol procedure for fetuin isolation results in a molecule with an altered secondary structure interpreted in terms of a loss cf helix content and trypsin resistance (6) and an increase in effective volume (8). The stability of salt-prepared fetuin to proteolytic attack resembles that of the al-acid glycoprotein, orosomucoid, which, like fetuin, contains a high content of sialic acid, the removal of which results in proteolytic suscept.ibility (9). Optical rotatory dispersion data in the visible region suggested a low helical content for fetuin, in contrast to observations on the al-acid glycoprotein by Schmid and Kamiyama (10). Since p&al or essentially complete removal of sialic acid from fetuin results in an apparent loss of secondary structure, on the basis of visible optical rotatory dispersion measurements, it was felt desirable to confirm this observation by ultraviolet optical rotatory measurement.s as well. In addition, the effect of the removal of sialic acid on the molecular weight and tryptic susceptibility of fetuin has been investigated. The association reaction of fetuin in the pH region near its isoelectric point (8) has been studied in more detail as well, along