The Action of Papain on Beef Serum Pseudoglobulin and on Diphtheria Antitoxin

It is generally believed that the proteolytic action of papain differs from that of enzymes of animal origin such as pepsin and trypsin. Annetts (1) found that when egg albumin is treated with papain-cyanide at pH 5 the course of digestion is marked not only by a progressive increase in material of low molecular weight but also by changes in the remaining albumin molecules. From ultracentrifugal and electrophoretic evidence it appeared that no unchanged egg albumin remained in the digests. In contrast to this, Tiselius and Eriksson-Quensel (20) showed that during the digestion of egg albumin by pepsin the remaining heavy protein retains the physical and immunological properties of native egg albumin, even when a considerable fraction of the albumin has been split into fragments of low molecular weight. Lundgren (6) has shown that crystalline papain catalyzes the denaturation of thyroglobulin prior to hydrolytic fissure of the protein. Papain also inactivates typhoid agglutinins which are resistant to pepsin and trypsin (16), and ragweed pollen extracts resistant to pepsin, trypsin, and erepsin (7). Whereas pepsin acts at hydrogen ion concentrations at which its substrate is positively charged, and trypsin on negatively charged proteins (8), papain has its pH optimum at 5.0, near the isoelectric point of most proteins (21). In view of the destructive action of papain on other proteins, the finding by Pope (14) that active horse diphtheria antitoxin can be recovered from antibody treated with papain, as well as with pepsin and trypsin, is surprising. It seemed of interest to determine whether this protein was unique in its resistance to the enzyme, or whether the action of papain might be less severe than has generally been thought. An ultracentrifugal analysis of the action of papain on beef serum pseudoglobulin and on diphtheria antitoxin has therefore been made. These proteins have the advantage, as shown previously (11, 12), that their primary and secondary cleavage products are still large enough to retain their protein characteristics, and to sediment easily. In the case of the antitoxin Pope’s finding has been confirmed and the sedimentation properties and flocculation behavior of the digests have been studied. Materials-The beef serum pseudoglobulin was prepared by ammonium sulfate fractionation and dialysis, as previously described (11).