Selective condensation of the unprotected fragments of a-globin-namely, a1..30 and a31.141-is catalyzed by Staphylococcus aureus V8 protease in the presence of 25% 1-propanol. The propensity of 1-propanol to induce the ahelical conformation and to generate a "native-like" topology for the polypeptide chain has been now investigated in an attempt to understand the molecular basis of this enzymecatalyzed stereospecific condensation. Removal of heme from the a-chain decreases the overall a-helical conformation of the protein considerably. A significant amount of the a-helical conformation is restored in the presence of25% 1-propanol and the digestion of a-globin by V8 protease becomes more selective concomitant with the increase in helicity. V8 protease digestion of a-globin at pH 6.0 and 40C occurs at Glu-30, Asp47, Glu-27, and Glu-23 in the absence of 1-propanol. In the presence of 25% 1-propanol, the digestion is selective to the peptide bond of Glu-30. This selectivity appears to be a characteristic feature of the native conformation of a-chain (polypeptide chain with bound heme). 1-Propanol induces the a-helical conformation into RNase S. peptide also. However, this increased helical conformation did not protect the RNase S peptide from V8 protease digestion at the Glu-9-Arg-10 peptide bond. RNase S peptide is in an a-helical conformation in RNase S, an interacting fragment-complementing system of S protein and S peptide. S peptide is resistant to V8 protease hydrolysis in this conformation. Thus, the resistance of a peptide bond in a segment of a protein to protease digestion appears to be a consequence of the secondary structure as well as the tertiary interactions of this segment with the rest of the molecule. The results suggest that the 1-propanol induces a-helical conformation into segments of a-globin as well as packing of these helices in a native-like topology.There has been considerable interest in recent years in the structure of proteins in organic solvents (1). The lowtemperature studies of enzymes, cryoenzymology, as well as low-temperature crystallography have necessitated the use of organic solvents in these protein structural studies (2). In addition, many enzyme-catalyzed reactions are also being investigated in organic media to enhance the industrial applications, specifically to take advantage of the high selectivity and specificity of the enzyme (3). With proteases, it has been possible to achieve "reverse proteolysis"-i.e., use proteases to catalyze the synthesis of peptide bonds by incorporating large amounts of organic solvents (i.e., like ethylene glycol, glycerol, etc.) in the reaction mixtures (4). The reformation of ribonuclease A from RNase S (5) and nuclease A from nuclease T (6) are the classical examples of this reverse proteolysis approach for semisynthesis of proteins (7).The protease-catalyzed reverse proteolysis for the semisynthesis of noncovalent analogues of proteins has been emerging as a useful procedure to prepare covalent variants of proteins (7). The presence...