Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy

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Abstract
Ca titration of the conformational change in bovine cardiac and rabbit skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2-7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+-binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for Ca of 1.8 .times. 107 and 4.5 .times. 105 M-1 were determined for the 2 classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (.apprx. 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent NMR study on this system. With the cardiac protein at pH 7.07, an apparent affinity constant for Ca of 2.0 .times. 107 M-1 was calculated while no low-affinity site at this pH was detected by CD. At lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+-binding site is detected with an apparent binding constant of 3.7 .+-. 104 M-1.