Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules.
Ca2+ titrations of the intrinsic fluorescence of a series of gamma-carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibit Tm (the (Ca2+)total concentration at which ln (B/F) = 0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase in Tm to 5.4 mM is observed for 8-GLA fragment 1. Tm decreases to 3.8 mM for the 7- and 6-GLA proteins. The value of h, about 2.8 +/- 0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2-1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca2+, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes in Tm and h response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.
Malhotra
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Medical Research Service, Department of Veterans Affairs, Cleveland, Ohio.
Valencic
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Fossel
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Koehler
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D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology*; D004586:Electrophoresis; D005453:Fluorescence; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry*; D011516:Prothrombin / Q000032:analysis / Q000737:chemistry*
D010446:Peptide Fragments; D011498:Protein Precursors; D015055:1-Carboxyglutamic Acid; C029733:prothrombin fragment 1; D011516:Prothrombin; D002118:Calcium