Dansyl Fluorescence Research Articles

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.

Human High Density Lipoprotein, Apolipoprotein Glutamine II: THE IMMUNOCHEMICAL AND LIPID-BINDING PROPERTIES OF APOLIPOPROTEIN GLUTAMINE II DERIVATIVES

Abstract Apolipoprotein glutamine II (apoLP-Gln-II) is one of the major protein constituents of human plasma high density lipoproteins. The protein is of known amino acid sequence and contains two identical polypeptide chains of 77 amino acids, linked by a single disulfide bridge at residue 6. We have investigated the effects of chemical modification of this disulfide linkage on the physical, immunological, and lipid-binding properties of the protein. ApoLP-Gln-II was reduced and alkylated with three reagents which are selective for the sulfhydryl linkage: (a) iodoacetic acid; (b) the spin label, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)bromoacetamide (TBA); and (c) a fluorescent label, 1-dansyl-2-maleimido ethylenediamine (DME). The three alkylated derivatives were isolated and chemically characterized. It was determined that 0.9 mole of carboxymethylcysteine, 0.8 mole of TBA, and 0.8 mole of DME were present per mole of protein monomer. ApoLP-Gln-II, Cm-apoLP-Gln-II, and TBA-apoLP-Gln-II were reconstituted with phosphatidylcholine. The protein-phospholipid complexes were isolated by ultracentrifugation in sucrose density gradients. The complexes contained, respectively, 1.23, 1.18, and 0.95 mg of phosphatidylcholine per mg of protein, representing approximately 13 moles of phosphatidylcholine per mole of protein. When ribonuclease was reduced and alkylated with iodoacetic acid, incubated with phosphatidylcholine, and subjected to density gradient centrifugation in a way identical with apoLP-Gln-II, the isolated ribonuclease contained less than 0.05 mg of phosphatidylcholine per mg of protein. The helicity of apoLP-Gln-II was reduced from approximately 46 to 37% by reduction and alkylation. This reduction could be reversed by reconstitution with phosphatidylcholine. The helicity of apoLP-Gln-II was increased from 46 to 61% and Cm-apoLP-Gln-II from 37 to 56% by reconstitution with phosphatidylcholine. Similar results were obtained with TBA-apoLP-Gln-II. By contrast, circular dichroism measurements indicated no increase in the α helical content of Cm-RNase when the reconstitution with phosphatidylcholine was performed as with apoLP-Gln-II. The electron paramagnetic resonance spectrum of TBA-apoLP-Gln-II indicated a rapidly tumbling spin label with a high degree of rotational freedom. The mobility of the spin label was not significantly altered by reconstitution with either phosphatidylcholine or with phosphatidylcholine plus cholesteryl oleate. A characteristic fluorescent spectrum of the dansyl chromophore was obtained with DME-apoLP-Gln-II. Reconstitution with phosphatidylcholine did not produce any significant change in the position or intensity of the dansyl fluorescence. ApoLP-Gln-II and each of the alkylated derivatives gave precipitin lines of complete identity with antisera prepared against apoLP-Gln-II. No immunological changes were detected after reconstitution of the apoprotein or its alkylated derivatives with phosphatidylcholine. Our findings show that, while reduction of the disulfide linkage of apoLP-Gln-II may affect the secondary structure of the protein, this effect is seen only in the absence of lipid. The integrity of the disulfide bond does not appear to be critical for binding of phosphatidylcholine. As assessed by EPR and by fluorescent labels selective for the sulfhydryl group, the environment of apoLP-Gln-II at or near residue 6 does not appear to be significantly altered by the interaction with and binding of phosphatidylcholine by the protein.