Conjugates of catecholamines. VII. Synthesis and beta-adrenergic activity of peptide catecholamine conjugates

A series of novel catecholamine derivatives has been prepared in which one of the N-methyl substituents of isoproterenol has been extended by a spacer consisting of a chain of four methylenes which terminates with an amide linkage to a peptide, the point of attachment being via the aromatic amino group of p-aminophenylalanine. In one of the derivatives, two catecholamines are attached to the same peptide in this manner. The peptides, which range in size from three to eight amino acid residues and contain phenylalanine, glycine, and L-alpha-amino-delta-hydroxyvaleric acid, were synthesized via stepwise and fragment condensation techniques. The beta-adrenergic agonist activities of the derivatives were evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells.     

Fluorescence properties of o-phthaldialdehyde derivatives of amino acids.

The fluorescence properties of the products formed by reaction of o-phthaldialdehyde with amino acids and their derivatives, in the presence of thiol compounds, have been studied. The emission spectra, quantum yields, and lifetimes depend on the primary amine and thiol compound used; the observations confirm the report (Simsons, S.S., Jr. and Johnson, D.F. (1978) J. Org. Chem 43, 2886--2891) that the product incorporates molecules of all three types of compounds. The fluorescence quantum yields of o-phthaldialdehyde derivatives of the naturally occuring amino acids ranged from 0.33 to 0.47, using 2-mercaptoethanol as the thiol compound. The fluorescence lifetimes were about 18--20 ns. Lower quantum yields were obtained when mercaptoethanol was replaced by dithiothreitol or ethnethiol. Derivatives of amino acid amides and peptides had quantum yeilds as low as 0.03, due to quenching by the carboxamide group. The intramolecular quenching was relieved by the detergent, sodium dodecyl sulfate, and by dimethylsulfoxide. Monosubstituted lysine exhibited a normal fluorescence, but the di-substituted product was largely quenched, presumably due to interaction between the two isoindole fluorophors. Fluorscence stopped-flow experiments showed that the alpha- and epsilon-amino groups reacted at different rates, with the epsilon-amion group reacting 10 times faster, with a t 1/2 of about 6 s under pseudo first order conditions at pH 9.0 with 10(-3) M o-phthaldialdehyde. The amount of instability shown by the o-phthaldialdehyde derivatives depended on the thiol compound used, the primary amine involved, and the solvent. Cysteine and o-phthaldialdehyde reacted to give an unstable, weakly fluorescent product; but cysteine could be assayed normally if its sulfhydryl was blocked. The o-phthaldialdehyde reagent was discussed in relation to fluorescamine, another reagent for primary amines.     

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.     

Aromatic phosphonates inhibit the lysophospholipase D activity of autotaxin

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. c Log P calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo.     

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

Perylene attached to 2'-amino-LNA: synthesis, incorporation into oligonucleotides, and remarkable fluorescence properties in vitro and in cell culture

During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in vitro and in vivo. Herein, the synthesis of 2'- N-(perylen-3-yl)carbonyl-2'-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied 'zipper' motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields Phi F = 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to Phi F increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2'-position of 2'-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in vivo providing excitation wavelength, which completely eliminates cell autofluorescence.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

193 nm photolysis of aromatic and aliphatic dipeptides in aqueous solution: dependence of decomposition quantum yield on the amino acid sequence

The values of molar absorption coefficient and quantum yields of photodecomposition and peptide bond scission were determined for a number of aromatic and aliphatic dipeptides under 193 nm laser irradiation in neutral argon-saturated aqueous solution. Under these conditions we could show that no dependence of the dipeptide decomposition quantum yield on the sequence of amino acid residues exists, neither for aromatic dipeptides nor for aliphatic ones.     

Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent $\text{p}\text{K}$ of the terminal carboxyl group of Lys-Trp-Lys is raised by ～3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.     

Stereospecific binding of diastereomeric peptides to salmon sperm deoxyribonucleic acid. Further evidence for partial intercalation

A series of diastereomeric dipeptide amides, containing an N-terminal L-lysyl residue and a C-terminal L- or D-amino acid with a derivatized aromatic ring on the side chain, was synthesized to determine the dependence of (1) the chirality of the N-terminal amino acid alpha-carbon and (2) the length of the N-terminal amino acid side chain for intercalation of the aromatic ring. The nature of the complex between the peptide and DNA (i.e., electrostatic, intercalative, or a combination of these) was determined by UV and CD studies, viscometric titrations, and 1H NMR studies. The results of these studies reveal distinct differences in the binding site of the aromatic rings of the various peptides. In particular, the results suggest that the alpha- and epsilon-amino groups of the lysyl residue bind electrostatically to adjacent phosphates on the DNA backbone in a stereospecific manner. As a result of this stereospecificity, the aromatic rings of the peptides with the L-L designation point toward the DNA helix, while those of the peptides of the L-D designation point away from the helix. This is completely consistent with previously reported work [Gabbay, E.J., Adawadkar, P. D., & Wilson, W. D. (1976) Biochemistry 15, 146; Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S., & Wilson, W. D. (1976) Biochemistry 15, 152]. The results also indicate a great dependence on the length of the side chain for intercalation of the aromatic ring. Specifically, if the side chain is long enough, and flexible enough, the aromatic ring can fully or partially intercalate, regardless of the chirality of the N-terminal amino acid alpha-carbon. However, if the side chain is too short, only partial intercalation is observed for peptides of the L-D designation, and no intercalation is observed for peptides of the L-D designation.     

Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.     

Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores.

The spectroscopic properties of a new series of fatty acid analogs in which a dipyrrometheneboron difluoride fluorophore forms a segment of the acyl methylene chain are presented and their characteristics as fluorescent membrane probes are examined. When incorporated as a low mole fraction component in model phospholipid membranes, the probes retain the principal characteristics of the parent fluorophore: green fluorescence emission with high quantum yield, extensive spectral overlap, and low environmental sensitivity. The fluorescence quantum yield is typically two to three times that of comparable membrane probes based on the nitrobenzoxadiazole fluorophore. The spectral overlap results in a calculated F?rster energy transfer radius (Ro) of about 57 A. Consequently, increasing fluorescence depolarization and quenching are observed as the mole fraction of the probe species incorporated in the membrane is increased. Low environmental sensitivity is manifested by retention of high quantum yield emission in aqueous dispersions of fatty acids. Partition coefficient data derived from fluorescence anisotropy measurements and iodide quenching experiments indicate that in the presence of fluid phase phospholipid bilayers the aqueous fraction of fatty acid is very small. Fluorescence intensity and anisotropy responses to phospholipid phase transitions are examined and found to be indicative of nonrandom fluorophore distribution in the gel phase. It is concluded that the spectroscopic properties of the fatty acid probes and their phospholipid derivatives are particularly suited to applications in fluorescence imaging of cellular lipid distribution and membrane level studies of lateral lipid segregation.     

Aromatic interactions in homeodomains contribute to the low quantum yield of a conserved, buried tryptophan

Trp 48, a conserved, buried residue commonly found in the hydrophobic core of homeodomains, has an unusually low fluorescence quantum yield. Chemical denaturation of Drosophila homeodomains Engrailed and Antennapedia(C39S) result in a four-fold increase in quantum yield, while unfolding of Ultrabithorax causes a twenty-fold enhancement. Global analysis of time-resolved fluorescence decay monitored at multiple emission wavelengths reveals sub-nanosecond lifetime components which dominate the overall intensity. Based on structure and sequence analysis of several homeodomains, we deduce that quenching is due to a transient, excited-state NH ellipsis pi hydrogen bond involving Trp 48 and a conserved aromatic residue at position 8. Additionally, both time-resolved fluorescence of indole-benzene mixtures and an electrostatic model of the proposed tryptophan-aromatic interaction substantiate different aspects of this mechanism. A survey of the Protein Data Bank reveals many proteins with tryptophan-aromatic pairs where the indole nitrogen participates in a NH ellipsis pi hydrogen bond with the ring of another aromatic residue. Chemical denaturation of one protein found in this survey, human fibronectin type III module 10, causes an enhancement of the fluorescence quantum yield. This unique interaction has implications for many other systems and may be useful for studying larger, multi-tryptophan containing proteins.     

Spectral, photophysical and photochemical properties of tetra- and octaglycosylated zinc phthalocyanines

Photophysical and photochemical properties of a series of tetra- and octaglycosylated zinc phthalocyanines (ZnPcs) substituted with glucose and galactose moieties have been reported. Spectral properties of these phthalocyanines are compared in DMSO. Absorption spectra of the non-peripherally tetra-substituted ZnPcs 2 showed a significant red shift in their Q-band maxima as compared to the peripherally substituted analog 1. All the complexes gave high triplet quantum yields ranging from 0.68 to 0.88, whereas triplet lifetimes were in the range of 100-430 μs in argon-saturated solutions. The octagalactosylated ZnPc 3b showed the highest triplet quantum yield and singlet oxygen quantum yield of 0.88 and 0.69, respectively. The fluorescence quantum yields and lifetimes of all the compounds under investigation were within the range of zinc phthalocyanine complexes.     

Interaction of lipophilic quinones with membrane fragments of Paracoccus denitrificans and Staphylococcus epidermidis.

In quinone-depleted mitochondrial and Paracoccus denitrificans membranes the quantum yield of fluorescence of ostruthin (6-geranyl-7-hydroxycoumarin) was maintained, whereas an increase in the quantum yield took place after extraction of Staphylococcus epidermidis membrane. A marked quenching effect of ubiquinone and menaquinone each with two isoprene units in the side chain on the ostruthin fluorescence was found with all types of quinone-depleted particles. When the homogues of menaquinone and ubiquinone with six isoprene units in the side chain were re-incorporated, a quenching of the ostruthin fluorescence was observed in the S. epidermidis membranes but not in those of P. denitrificans. The different behaviour of both bacterial preparations is attributable to the more specific finding of ubiquinone in the particles of P. denitrificans.     

Pulsefluorimetry of tyrosyl peptides

The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x₁ which determines the conformation of the tyrosyl residue around its C_α-C_β bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

Fatty acid binding sites of rodent adipocyte and heart fatty acid binding proteins: characterization using fluorescent fatty acids

Murine adipocyte and rat heart fatty acid binding proteins (FABP) are closely related members of a family of cytosolic proteins which bind long-chain free fatty acids (ffa). The physical and chemical characteristics of the fatty acid binding sites of these proteins were studied using a series of fluorescent analogues of stearic acid (18:0) with an anthracene moiety covalently attached at seven different positions along the length of the hydrocarbon chain (AOffa). Previously, we used these probes to investigate the binding site of rat liver FABP (L-FABP) [Storch et al. (1989) J. Biol. Chem. 264, 8708-8713]. Here we extend those studies to adipocyte and heart FABP, two members of the FABP family which share a high degree of sequence homology with each other (62% identity) but which are less homologous with L-FABP (approximately 30%). The results show that the fluorescence emission spectra of AOffa bound to adipocyte FABP (A-FABP) are blue-shifted relative to heart FABP (H-FABP), indicating that AOffa bound to A-FABP are held in a more constrained configuration. For both proteins, constraint on the bound ffa probe is highest at the midportion of the acyl chain. Ffa are bound in a hydrophobic environment in both proteins. Excited-state lifetimes and fluorescence quantum yields suggest that the binding site of H-FABP is more hydrophobic than that of A-FABP. Nevertheless, acrylamide quenching experiments indicate that ffa bound to H-FABP are more accessible to the aqueous environment than are A-FABP-bound ffa.(ABSTRACT TRUNCATED AT 250 WORDS)