Optical Rotatory Dispersion and Circular Dichroism of Pepsinogen, Carbamylpepsinogen, and Succinylpepsinogen

Abstract

Abstract Studies of optical rotatory dispersion (ORD) and circular dichroism (CD) are reported on pepsinogen, carbamyl-, and succinylpepsinogen. From the ORD measurements in the wave length range of 600 to 300 mµ as functions of urea, temperature, and pH, it was shown that the macromolecular conformation of the three proteins differs markedly. On replacing the e-NH2 groups of the lysine residues of pepsinogen by non-polar groups (carbamylpepsinogen) or acidic side chains (succinylpepsinogen), the electrostatic side chain interaction between the basic residues and some dicarboxylic acids which stabilizes pepsinogen is abolished. With all three proteins the changes in [η], particularly when measured as function of pH, parallel those of the specific rotation, [α]366. The ORD spectra below 300 mµ show several peaks and troughs. The principal trough of the three proteins in phosphate buffer of pH 7.7 and 0.1 ionic strength is at 227 mµ and is shifted to 230 mµ if the temperature is raised or the pH of the solutions is altered. The positive maximum is at 202 mµ for pepsinogen, at 200 mµ for carbamylpepsinogen, and at 195 mµ for succinylpepsinogen. The ORD spectra above 260 mµ show a trough at 278 mµ, most likely involving the aromatic side chains. These patterns are very different from those characteristic for α-helical structures. The CD spectra of the three proteins at pH 7.7 are characterized by a strong negative band at 212, 205, and 202 mµ for pepsinogen, the carbamyl, and succinyl derivative, respectively. These are displaced to lower wave lengths in the alkaline pH range. The positive CD band of pepsinogen in the phosphate buffer of pH 7.7 is at 193 mµ. Above 250 mµ, dichroic bands were recorded at 255, 264, 278, 292, and 300 mµ. The band at 278 mµ is pH-dependent and was assigned to the aromatic amino acid residues. As the pH is raised above pH 10.0, the band passes through zero and changes sign. ORD spectra have been calculated from the CD data for pepsinogen, carbamyl-, and succinylpepsinogen by the use of a Kronig-Kramers transform with a computer program. Comparison with the observed ORD data shows excellent agreement with the patterns of the observed troughs and peaks. These results are discussed in the light of ORD and CD spectra of known conformations.