Folding analysis of hormonal polypeptide calcitonins and the oxidized calcitonins using electrospray ionization mass spectrometry combined with H/D exchange.
Conformational change of calcitonins has been examined by measuring the rate of hydrogen/deuterium (H/D) exchange in amino acids. Time dependent m/z shift caused by H/D exchange was monitored by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT MS). The rate constants of H/D exchange were calculated from apparent first-order kinetics. The time course of H/D exchange exhibited two phases of faster and slower exchange. The smaller rate constant (k2) estimated from the slower H/D exchange was correlated with an alpha-helix content that reflected the folding state. The order of k2 values obtained for human calcitonin (hCT), porcine calcitonin (pCT), salmon calcitonin (sCT), and elcatonin (ECT) was hCT > pCT approximately sCT > ECT. Although the amino acid sequence of sCT is similar to that of ECT, their k2 values were considerably different. The results suggest that ECT is relatively rigid on the N-terminal side cyclic structure in the folded state. Further, the effect of methionine oxidation on k2 has been examined. In the oxidized pCT that possesses similar biological activity with the intact pCT, the k2 values obtained were nearly equal. The k2 of hCT increased via methionine oxidation, and the biological activity was weakened by oxidation. This suggested that methionine oxidation of hCT produced unfolding in the secondary structure and that oxidative unfolding of hCT led to the loss of biological activity. The results indicate that the H/D exchange rate constant may be used as an informative parameter to elucidate the relationship between the folded state and biological activity of polypeptides like calcitonins with secondary structure.
Nabuchi
Yoshiaki
Y
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Gotemba, Japan. nabuchiysa@chugai-pharm.co.jp
Asoh
Yoshinori
Y
Takayama
Mitsuo
M
D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000031:analogs & derivatives* / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D012474:Salmon; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine
D006859:Hydrogen; D002116:Calcitonin; D003903:Deuterium; C012755:elcatonin