5F-BAPTA

Fig. 11. Changes In gated NMR spectra during the cardiac cycle. Top panel isovolumic left ventricular pressure In a ferret heart paced at 0.99 Hz in 8 mM [Ca +]. NMR spectra were acquired at the two times indicated on the pressure record (a) 10 ms prior to stimulation (b) 75 ms after stimulation. Middle panel shows gated F NMR spectra (each from 800 acquisitions) recorded at (a) and (b), as indicated. The bound (B) and free (F) peaks of 5F-BAPTA exhibit distinct chemical shifts at approximately 8 and 2 ppm, respectively, downfield from a standard of 1 mM 6-Ftryptophan at 0 ppm. It appears that the free [Ca +] varied during the cardiac cycle. Bottom panel shows gated P spectra (400 scans) acquired at times a and b in the same heart. The major peaks correspond to phosphocreatine (0 ppm), ATP (the three peaks upfield from phosphocreatine), and inorganic phosphate (the small peak at 4-5 ppm) (Reproduced from Marban et al. Circ. Res. 1988 63 673-678 [311] with permission of Lippincott, Williams Wilkins).

Multinuclear MRS studies in the IPRK with Na, P and Rb (a congener of potassium) MRS have demonstrated that increases in intracellular sodium and decreases in potassium accompany the decrease in ATP induced by hypoxia [331]. Multinuclear studies with F, 1, P and single, double and triple quantum Na MR have also been performed in IPRK by the Gupta group. Brief (10 min) ischemia in an IPRK loaded with the membrane-impermeant intracellular calcium indicator, 5F-BAPTA, caused a partially reversible in-... 

A series of symmetrically substituted fluorine derivatives of BAPTA (see Figure 3.3A) has been synthesized.One of these chelators is 5F-BAPTA (Figure 3.5A), which has a binding constant for Ca, of 1.4 x 10 M and a F NMR chemical shift, 8, that in the free ligand is different from that in the complex with Ca + (ASca2+ 6 ppm). The rate of Ca + dissociation, koft, is 5.7 X 10 s , which gives the rate of association, kon, as 8 x 10 M " s according to... 

A) Molecular structure of the calcium chelator 5F-BAPTA whose F chemical shift changes upon calcium binding. (B) F NMR spectrum of a solution containing 5F-BAPTA and Ca in a molar ratio of 3 1. Signal B originates from the Ca +-5F-BAPTA complex, and F from free 5F-BAPTA. Adapted from Reference 29. 

An additional beneficial property of 5F-BAPTA and other fluorinated analogues of BAPTA is that they will also bind other metal ions with a chemical shift of the complex that is characteristic of the metal ion. Under favorable circumstances, it is thus possible to measure simultaneously the concentrations of several cations. 

Intracellular Ca + has been measured by F NMR spectroscopy of intact hearts loaded with the 5,5 -di-fluoro derivative of l,2-bis(o-aminophenoxy)ethane-N,N,N, N -tetraacetic acid (5F-BAPTA) (Figure 5). 5F-BAPTA is loaded into the heart as the cell-per-meant acetoxymethyl (AM) ester. Esterases within the cardiomyocyte hydrolyse the AM ester to the free acid, which, being charged and therefore unable to cross the cell membrane, is trapped within the cell. The calcium-bound and calcium-free 5F-BAPTA species undergo slow exchange resulting in two NMR-visible peaks. The intracellular Ca concentration is... 

Animal studies have shown that in vivo F MRS can be used in metabolic studies. It has been shown that 3-fluoro-3-deoxy-D-glucose in the brain is metabolized primarily in the aldose reductase sorbitol pathway, suggesting that F MRS may be a useful tool for the further elucidation of this pathway. Further studies have shown that it is also possible to study the metabolism of fluorinated galactose, tryptophan and protein in vivo and determine intracellular calcium levels. The concentration of this cation was shown to be the same in the kidney, spleen and brain of rats, at approximately 200 nM. These measurements were obtained by infusing the calcium indicator 5F-BAPTA (=5,5-difluoro-l,2-bis(o-amino-phenoxy)ethane-N, N, M, A/ -tetraacetic acid) intravenously or intraventricularly into the animal, and so the potential application to human subjects appears limited. 

Most widely used methods are based on (i) the competition of protons with the bound metal ions for thiolate ligands, i.e., the pH stability of the metal-thiolate clusters or (ii) the competition with a metal chelator. In the former case the pH stability of the Cd-thiolate clusters is followed by absorption spectroscopy of the CysS-Cd(II) LMCT band at 250 nm. From the apparent p/Ca values the apparent stability constants of the clusters can be derived [103]. The apparent p/sTa values are determined either by taking the pH values of half-maximum absorbance or using a non-linear curve fit of the pH plot [104]. The other method is based on the competition for a single metal ion between the chelator 5F-BAPTA (1,2-bis-(2-amino-5-fluorophenoxy)ethane-A,W,A ,A -tetraacetic acid) and the protein followed by NMR spectroscopy [71]. Although this method was established for the zinc metalloforms of MTs, its applicability to cadmium metalloforms has also been demonstrated [105]. In this case, by analogy with zinc finger proteins a lower affinity for mixed Cys/His coordination of Cd(II) in MTs compared to sole Cys coordination has been shown. 

F-BAPTA 1,2 -bis-(2-amino-5-fluorophenoxy)ethane-fV, N, N -tetraacetic acid... 

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