The β/α Peak Height Ratio of ATP

From 31P NMR measurements made in vitro at 38°C, I = 0.25, pH 5.75-8.5, and calculated free [Mg2+] from 0 to 5 m, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 m, the chemical shift difference between the α- and β-ATP resonances, γα- and β, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, γPi, and the peak height ratio of the β- and α-ATP resonances, hβ- and α, could be related to free [Mg2+] by simultaneous solution of: pH=pKaH2PO4-+log(δPi−δH2PO4-)(δHP42-−δPi)+[Mg2+]KDMgHPO4(δMgHPO−δPi)[Mg2+]=2.5×KDMgATP2−×(1+10pKat−pH)×hβ/αhβ/αMax−hβ/α(Eq. 1) We found that hβ/α changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 m, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 ± 0.1 from hβ/α and 1.2 ± 0.03 from measured [citrate]/[isocitrate] but 0.51 ± 0.1 from γαβ. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hβ/α to 0.61 ± 0.02 and 0.74 ± 0.11 by [citrate]/[isocitrate] but the estimate from γαβ was unchanged at 0.46 ± 0.04 m. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [ΣADP]. From 31P NMR measurements made in vitro at 38°C, I = 0.25, pH 5.75-8.5, and calculated free [Mg2+] from 0 to 5 m, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 m, the chemical shift difference between the α- and β-ATP resonances, γα- and β, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, γPi, and the peak height ratio of the β- and α-ATP resonances, hβ- and α, could be related to free [Mg2+] by simultaneous solution of: pH=pKaH2PO4-+log(δPi−δH2PO4-)(δHP42-−δPi)+[Mg2+]KDMgHPO4(δMgHPO−δPi)[Mg2+]=2.5×KDMgATP2−×(1+10pKat−pH)×hβ/αhβ/αMax−hβ/α(Eq. 1) We found that hβ/α changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 m, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 ± 0.1 from hβ/α and 1.2 ± 0.03 from measured [citrate]/[isocitrate] but 0.51 ± 0.1 from γαβ. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hβ/α to 0.61 ± 0.02 and 0.74 ± 0.11 by [citrate]/[isocitrate] but the estimate from γαβ was unchanged at 0.46 ± 0.04 m. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [ΣADP].