The interaction of Ca²⁺‐binding proteins with the carbocyanine dye stains‐all

Abstract

SynopsisThe interaction of the carbocyanine dye Stains-all with the Ca2+-binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca2+, complexes with Stains-all of all three proteins exhibit at high dye : protein mole ratios an intense J absorption band at 600-650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J-band is progressively lost as the dye : protein ratio decreases and is replaced by bands of the y and P types at 450-550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J-band is observed; its intensity is undiminished at the lowest dye :protein ratios examined. In the presence of excess Ca2+ the J-band is lost for all three proteins. For calmodulin and troponin C it is replaced by yand P-bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.

N-terminal Proline-rich Domain Is Required for Scrambling Activity of Human Phospholipid Scramblases

“…Calcium Binding to hPLSCR1 and -2 and Other Mutant Proteins-Sequence analysis of hPLSCR1 and -2 revealed the presence of a single putative EF-hand-like Ca 2ϩ binding motif, which was confirmed using Stains-All dye (27). Binding of this dye to EF-hand-containing proteins produces a distinct peak at 650 nm termed as the J-band.…”

“…Stains-All was dissolved in 2 mM MOPS buffer (pH 7.2) containing 30% ethylene glycol. Stains-All produces a series of discrete spectra depending upon interaction and conformation of binding region (27). The free form of the dye produces two distinctive spectra at 535 nm (␤-band) and 575 nm (␣-band) that correspond to the monomeric and dimeric form of the dye.…”

Modified Helix‐Loop‐Helix Motifs of Calmodulin

“…All peptides and dye solutions were made in 2 mM Mops, pH 7.2, containing 30% ethylene glycol as described earlier (Caday and Steiner, 1985). The CD spectra were recorded on a Jasco 5-20 spectropolarimeter at room temperature.…”

“…Spectroscopic methods involving calcium-ion mimics such as lanthanide binding, or dye binding, are therefore used. The use of the cationic carbocyanine dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-Y-methyl-thiacarbocyanine bromide) to monitor calcium-ion binding to calcium-binding peptides and proteins has been established (Caday et al, 1986;Sharma et al, 1989Sharma et al, , 1993. This dye binds at the calcium-binding site, from where it can be displaced upon the addition of excess calcium (Caday and Steiner, 1985).…”

“…The use of the cationic carbocyanine dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-Y-methyl-thiacarbocyanine bromide) to monitor calcium-ion binding to calcium-binding peptides and proteins has been established (Caday et al, 1986;Sharma et al, 1989Sharma et al, , 1993. This dye binds at the calcium-binding site, from where it can be displaced upon the addition of excess calcium (Caday and Steiner, 1985). This spectral assay is easy, accurate and conformation sensitive (Sharma and Balasubramanian, 1991).…”