Bromocresol Purple as a Probe for Structural Features of Fragmented Sarcoplasmic Reticulum

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The binding and spectral properties of bromocresol purple (BCP), an acidic dye, were used as probe to investigate structural features of fragmented sarcoplasmic reticulum (SR) of skeletal muscle.
The wavelengths of maximum absorption intensity (λ_max) of BCP were 589 and 604nm in Tris-HCl buffer (pH 8.8) and bound to SR, respectively. Since spectral shift of BCP to longer wavelengths (red shift) is caused by approach of the hydrophobic radical of an adjacent molecule, it was suggested that the red shift of BCP on SR reflected the hydrophobic nature of SR.
When the Ca²⁺ concentration was less than 10^-9M, ATP released BCP molecules from SR. The resulting increase of free BCP concentration in the reaction mixture shifted the total spectrum to shorter wavelengths (blue shift), though λ_max of the bound BCP did not change. The release of BCP after ATP addition was interpreted as a result of increase in the number of nucleophilic sites on SR. If Ca²⁺ was present, ATP addition enhanced the binding of BCP and caused a marked decrease in the absorption intensity of bound BCP, suggesting a decrease in the number of nucleophilic sites on SR during Ca²⁺ transport.
The blue shift caused by release of BCP was used to estimate the interaction of ATP with SR using a dual-wavelength spectrophotometer, yielding association constants of ATP for SR of 1.2×l0⁵ and 2.5×lO⁴M^-1 in the presence and absence of Mg, respectively.