Pepsin iodine

Iodination of PIR (147) showed 1 residue buried, Tyr 25, and all others iodinated at least to the monoiodotyrosyl form. Pepsin-inactivated RNase also has only one abnormal tyrosyl by titration which is thus assumed to be 25. Iodination of RNase-S is very similar to RNase-A in the early stages (lift). Extensive iodination leads to dissociation of the protein and peptide components. Direct iodination of S-protein indicated that all 6 tyrosyl residues were accessible, in this sense comparable to urea-denatured RNase-A. Substantial structural changes must be involved for both S-protein and PIR if Tyr 97, in particular, is to become susceptible to attack (see Section IV,B,3). 

Behaves like a globulin with a broad isoelectric zone. Biological activity is destroyed by heating and by proteolysis with pepsin, trypsin, and chymotrypsin. Also inactivated by oxidizing agents, such as potassium permanganate and elemental iodine. 

Careful acetylation of pepsin with ketene has yielded preparations with no free amino groups, but with full activity. More extensive acetylation inactivates the enzyme. Tyrosine side chains appear to be somewhat more important to the enzyme, but some activity remains after partial iodination, and treatment with nitrous acid leaves 50 per cent of the activity. 

The reviewer has been surprised at the high degree of homogeneity of certain reaction mixtures. When pepsin was treated with ketene (9) or iodine (7) under mild and controlled conditions the bulk of the reaction mixture was relatively uniform, as shown by fractionation procedmres designed to detect the presence of products with extremely different properties. This result may be interpreted as indicating that most of the groups of the type which react first, i.e., at the highest rate, are saturated before those of the next type have reacted appreciably. 

Although earlier workers iodinated proteins, Ostwald in 1910 provided the first unequivocal proof that tyrosine in proteins was iodinated. He isolated diiodotyrosine from iodinated albumin (273), gliadin (274), and casein (275)-. Since then papers on serum albumin, egg albunun, and serum globulin (276), pepsin (6, 7), insulin (125), and the lactogenic hormone (277) all indicate that the action of iodine on these proteins in neutral or slightly alkaline solution is one of substitution on the tyrosine residues. The reaction is as follows ... 

Crystalline mono iodotyrosine was also isolated from pepsin treated with a small quantity of iodine (7). Some of its properties did not agree with those of synthetic mono iodotyrosine so that its exact nature has been questioned (297). The isolation from peptin has been repeated and a comparison made with a synthetic product. Such properties as solu-> bility, ultraviolet absorption spectra, pK s, and distribution coefficients were compared and cross-examined wherever possible. There can be no doubt that the crystalline product from pepsin is mono iodotyrosine (298). 

Iodination of proteins has produced measurable changes in the titration curves. In zein (280), insulin (125), and pepsin (6) the region of the titration curves usually assigned to the phenolic group of tyrosine (pK=10) is displaced in iodinated proteins in the direction of increased acidity by nearly 2 pH units. This is apparently not true for iodinated globin (299). 

Rate measurements on pepsin and serum albumin have led Li (300) to conclude that all tyronnes in a ven protein molecule are not iodinated at the same rate. He also suggests that availability of groups in the protein may be a factor. The availability or reactivity of protein groups is receiving considerable attention. Anson (34) and Greenstein and Edsall (83) have discussed it in connection with SH determinations while Crammer and Neuberger (75), Cannan (74), Philpot and Small... 

From completely iodinated pepsin after alkaline hydrolysis and partial purification at least 82% of the total iodine was accounted for in a solution analyzing as diiodotyrosine (7). 53fo was actually crystallized. 

Iodinated serum albumin (301, 290) and iodinated pepsin (7) have been crystallized and some of their properties examined. In the latter instance the electrophoresis and solubility tests indicated a high degree of homogeneity yet it was clearly different from the original pepsin. 

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