BODIPY Groups Research Articles

Intramolecularly Quenched BODIPY-Labeled Phospholipid Analogs in Phospholipase A2 and Platelet-Activating Factor Acetylhydrolase Assays and in Vivo Fluorescence Imaging

Phospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A2 (cPLA2) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA2 (specific activities: 18 and 5 nmol min−1 mg−1) and all were good for rPAF-AH (specific activities: 17, 11, and 6 μmol min−1 mg−1). The minimal amount of enzyme detectable was 50 ng for cPLA2 and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA2 in zebrafish embryo extracts and one was well suited for imaging of PLA2 activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA2 activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.

The use of site-directed fluorophore labeling and donor–donor energy migration to investigate solution structure and dynamics in proteins

The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.

Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence quenching

The location of a series of lipophilic and lipid-attached BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene) membrane probes was analyzed by the quenching of BODIPY fluorescence by a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. When attached to the polar headgroup of PE the BODIPY remained near the polar headgroup in depth. However, when attached at the end of free or phospholipid-attached fatty acyl chains, or when attached to two hydrocarbon chains, we observed two probe populations. One, usually dominant, population of BODIPY groups ‘looped back’ towards the surface, but a second population remained deeply embedded within the bilayer. When attached to a fatty acid or fatty acyl chain, the deep population appeared to locate at a depth related to its point of attachment to the acyl chain. In BODIPY linked to free fatty acids, the location of the deep population responded to the ionization of the carboxyl group. Because, unlike NBD (7-nitro-2,1,3-benzoxadiazol-4-yl) and most dansyl groups, acyl chain linked BODIPY groups can exist in a deeply buried form we conclude that BODIPY linked acyl chains are superior to NBD or dansyl linked acyl chains as membrane probes.