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. 1972 Oct;12(10):1251–1265. doi: 10.1016/s0006-3495(72)86160-5

Analysis of a Calcium Ion Binding System Composed of Two Different Sites

Tsuyoshi Ohnishi, Edward J Masoro, Helen A Bertrand, Byung Pal Yu
PMCID: PMC1484220  PMID: 4672618

Abstract

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca++ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca++ binding sites and without such solute were titrated with Ca++ using Mx as an indicator of free Ca++ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca++; the association constants of the high affinity site and low affinity site are 4.3 × 105 M-1 and 9 × 103 M-1, respectively. The advantages and limitations of this methodology are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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