Segal J
Biotechnol Appl Biochem. 1986 Oct;8(5):423-9.
Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-calcium buffer is widely used in various calcium-dependent reactions where free calcium concentrations of 1 microM or less are desirable. The free calcium concentration is calculated from the association constant of EGTA . Ca2- and serves as the true available calcium in systems devoid of a constituent with high affinity to Ca2+ other than EGTA. But, it is conceivable that in systems with high affinity to Ca2+ (comparable to that of EGTA) this is not the case, because such systems will compete with EGTA for the total calcium content in the medium, so that the true available calcium for these systems is greater than that calculated from the EGTA buffer. This hypothesis was tested in three different Ca+-modulated systems: Quin 2 fluorescence, Ca2+-ATPase, and adenylate cyclase, in which the response of the system to calcium was compared between EGTA-free media, containing known amounts of added calcium, and the EGTA-Ca2+ buffer media. In all three systems, the amount of available calcium in the EGTA-Ca2+ buffer medium was much greater than the calculated free Ca2+ concentration. This indicates that in systems with high affinity to Ca2+, preparation of available Ca2+ in concentrations of 1 microM or less must account for both the EGTA and the system capacities for calcium.
乙二醇双（β-氨基乙醚）-N，N'-四乙酸（EGTA）-钙缓冲液广泛应用于各种钙依赖性反应中，这些反应需要1微摩尔或更低的游离钙浓度。游离钙浓度是根据EGTA·Ca²⁺的缔合常数计算得出的，并且在除EGTA外没有对Ca²⁺具有高亲和力成分的系统中，它代表真正可利用的钙。但是，可以想象，在对Ca²⁺具有高亲和力（与EGTA相当）的系统中情况并非如此，因为这样的系统会与EGTA竞争培养基中的总钙含量，以至于这些系统真正可利用的钙大于从EGTA缓冲液计算得出的量。该假设在三种不同的Ca⁺调节系统中进行了测试：喹啉2荧光、Ca²⁺-ATP酶和腺苷酸环化酶，在不含EGTA的培养基（含有已知添加钙量）和EGTA-Ca²⁺缓冲液培养基之间比较了系统对钙的反应。在所有这三种系统中，EGTA-Ca²⁺缓冲液培养基中可利用的钙量远大于计算出的游离Ca²⁺浓度。这表明，在对Ca²⁺具有高亲和力的系统中，制备浓度为1微摩尔或更低的可利用Ca²⁺时，必须同时考虑EGTA和系统对钙的容纳能力。
