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B P Maliwal

Publications and source records attributed to B P Maliwal.

At least 19 recordsLinked to original sources
PubMed1986

Resolution of complex anisotropy decays by variable frequency phase-modulation fluorometry: a stimulation study.

We used simulations to determine the resolution of complex anisotropy decay laws which is obtainable by frequency-domain fluorometry. The simulations include the effects of torsional and segmental motions of tryptophan residues in proteins, the multiple correlation times of asymmetric molecules, and three-component anisotropy decays. For a protein with a global correlation time of 10 ns it should be possible to resolve torsional motions with correlation times as short as 10 ps if the amplitude of the rapid motion is at least 20% of the total anisotropy decay with r0 = 0.4. Correlation times which differ by only 1.4-fold can be resolved, making this method useful for determination of the shape of proteins and other asymmetric molecules. It is possible to resolve three-component anisotropy decays if the overall difference among the correlation times is 30-fold. Such resolution will be useful for understanding of internal motions of proteins and membranes. The validity of these predictions is demonstrated in the subsequent paper using experimental data for melittin in solution and when bound to membranes (Maliwal, B.P., Hermetter, A. and Lakowicz, J.R. (1986) Biochim. Biophys. Acta 873, 173-181).

Amino Acids↗
PubMed1986

A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin.

Internal motions of melittin and its lipid complexes were studied by anisotropy decays determined by frequency-domain fluorometry. A covalent anthraniloyl probe was attached, probably to lysine-21. The emission spectra indicate that the anthraniloyl moiety is exposed to solvent in both monomeric and tetrameric forms and is present at the lipid-water interfacial region in the lipid complexes. The fluorescence intensity decay of melittin in solution and its lipid complexes was characterized by three lifetimes. The lifetimes were near 1-2 ns, 6-7 ns and 10 ns. At increased temperatures there was an increase in the amplitude of the intermediate lifetime and a decrease in that of the longer lifetime. For all the melittin systems, at least three correlation times were required to fit the anisotropy data. Of the three correlation times, the shortest correlation time represents the local motions of the probe, while the longest represents global motions of the whole molecule. The intermediate correlation time probably represents the dynamics of domains/helices within the molecule. The melittin monomer is highly flexible, with greater than 90% of its anisotropy being lost by the local motions. Even though it is well organized (greater than 75% helical), the tetramer is still a highly flexible molecule, with 70% of its anisotropy being lost by the local motions. The internal motions of melittin decrease upon binding to lipids and are sensitive to the phase state of the lipid complexes.

Bee Venoms↗
PubMed1986

Anchoring of phospholipase A2: the effect of anions and deuterated water, and the role of N-terminus region.

The effect of anions and deuterated water on the kinetics of action of pig pancreatic phospholipase A2 is examined to elaborate the role of ionic interactions in binding of the enzyme to the substrate interface. Anions and deuterated water have no significant effect on the hydrolysis of monomeric substrates. Hydrolysis of vesicles of DMPMe (ester) is completely inhibited in deuterated water. The shape of the reaction progress curve is altered in the presence of anions. The nature and magnitude of the effect of anions depends upon the nature of the substrate as well as of the anion. Substantial effects of anions on the reaction progress curve are observed even at concentrations below 0.1 M and the sequence of effectiveness for DMPMe vesicles is sulfate greater than chloride greater than thiocyanate. Apparently, anions in the aqueous phase bind to the enzyme, and thus compete with the anionic interface for binding to the enzyme. Binding of the enzyme to anionic groups on the interface results in activation and increased accessibility of the catalytic site possibly via hydrogen bonding network involving water molecule. In order to elaborate the role of the N-terminus region in interfacial anchoring, the action of several semisynthetic pancreatic phospholipase A2s is examined on vesicles of anionic and zwitterionic phospholipids. The first-order rate constant for the hydrolysis of DMPMe in the scooting mode by the various semisynthetic enzymes is in a narrow range: 0.7 +/- 0.15 per min for phospholipase A2 derived from pig pancreas and 0.8 +/- 0.4 per min for the enzymes derived from bovine pancreas. In all cases a maximum of about 4300 substrate molecules are hydrolyzed by each phospholipase A2 molecule. If anions are added at the end of the first-order reaction progress curve, a pseudo-zero-order reaction progress curve is observed due to an increased intervesicle exchange of the bound enzyme. These rates are found to be considerably different for different enzymes in which one or more amino acids in the N-terminus region have been substituted. Steady-state and fluorescence life-time data for these enzymes in water, 2H2O and in the presence of lipids is also reported. The kinetic and binding results are interpreted to suggest that the N-terminus region of phospholipase A2 along with some other cationic residues are involved in anchoring of phospholipase A2 to the interface, and the catalytically active enzyme in the interface is monomeric.

Amino Acid Sequence↗
PubMed1985

The environment of tryptophan in pig pancreatic phospholipase A2 bound to bilayers.

Binding of pig pancreatic phospholipase A2 to ternary codispersions of diacylphosphatidylcholine/lysophosphatidylcholine/fatty acid (100:22:22, mole ratio) is monitored by the increase in intrinsic fluorescence intensity of the single tryptophan residue. The fluorescence is quenched by the brominated fatty acid components in the ternary codispersions. The quenching efficiency is in the order: 11,12-dibromo- greater than 9,10-dibromo- greater than 6,7-dibromo- greater than 2-bromo fatty acid. The quenching efficiency of the 9,10-brominated derivatives of the three components in the ternary codispersions is in the order diacylphosphatidylcholine greater than fatty acid greater than lysophosphatidylcholine. Two isomers of diacylphosphatidylcholine with 9,10-dibromo substituents on chain 1 or 2 are equally efficient quenchers. While succinimide also quenches the fluorescence of the free and the membrane bound enzyme, the tryptophan residue in both systems is not accessible to 1-methylnicotinamide. These results are rationalized by a hypothesis that the acyl chains of the substrate interacts with the tryptophan residue of pig pancreatic phospholipase A2, which is readily accessible to water soluble neutral quenchers both in the free and the bound state.

Animals↗
PubMed1985

Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry.

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.

Benz(a)Anthracenes↗
PubMed1985

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization.

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.

Apolipoprotein C-I↗
PubMed1985

Construction and performance of a variable-frequency phase-modulation fluorometer.

We describe the construction and performance of a variable-frequency phase-modulation fluorometer. This instrument, which provides modulation frequencies from 1 to 200 MHz, was constructed using commercially available components. To facilitate the introduction of these instruments into other laboratories we describe in detail the chosen components and the principles of operation. The present light source is a continuous-wave helium-cadmium laser, which provides convenient excitation wavelengths of 325 and 442 nm. Modulation of the incident light is provided by one of several electro-optic modulators. The extent of modulation ranges from 1.0 to 0.2 as the frequency increases from 1 to 200 MHz. Phase angles and demodulation factors are measured using the cross-correlation method. The closely spaced frequencies are provided by two direct frequency synthesizers. The phase and modulation measurements are accurate to 0.2 degrees and 0.002, respectively, from 1 to 200 MHz. This accuracy allows considerable resolution of complex decay laws. The usefulness of frequency-domain fluorometry for the resolution of multiexponential decays is illustrated by the analysis of several difficult mixtures. As examples, we resolved a two-component mixture of anthracene (4.1 ns) and 9,10-diphenylanthracene (6.3 ns), and confirmed that the intensity decay of NADH in aqueous buffer is at least a double exponential (0.2 and 0.86 ns). We also resolved an especially difficult mixture of anthracene (4.1 ns) and 9-methylanthracene (4.5 ns), and a three-component mixture with decay times of 1.3, 4.1 and 7.7 ns. Frequency-domain fluorometers appear to be particularly useful for determination of complex decays of fluorescence anisotropy. This capability is illustrated by the determination of rotational correlation times as short as 47 ps for p-bis[2-(5-phenyloxazolyl)]benzene (POPOP) in hexane at 40 degrees C, and by the resolution of the two correlation times of anisotropic rotators such as perylene and 9-aminoacridine. Resolution of two anisotropy decay times for 9-aminoacridine is a difficult test because these correlation times differ by less than 2-fold. The resolution of multiexponential decays of intensity and anisotropy possible with this instrument is at least equivalent to that obtained using state-of-the-art time-resolved instruments based on mode-locked laser sources. The ease and rapidity of frequency-domain measurements, the relative simplicity of the equipment, the accuracy of the measurements and the lack of significant systematic errors indicate that frequency-domain fluorometry will be widely useful in chemical and biochemical research.

Aminoacridines↗
PubMed1985

Spectroscopic study of the interaction of gossypol with bovine serum albumin.

The interaction of gossypol with bovine serum albumin (BSA) at pH 7.6 in 0.02 M borax-borate buffer has been followed by circular dichroism (CD) and difference spectroscopy. From the extrinsic CD band at 390 nm, a binding constant of 2.7 X 10(3) M-1 was calculated. At 54 degrees the induced CD spectrum was abolished, suggesting that the interaction is not favoured at that temperature. The effect of various solvents and salts on the interaction has been followed by difference spectroscopy. The modification of epsilon-amino groups of lysine did not affect the interaction. Binding of gossypol to BSA does not cause a change in its secondary structure or sedimentation coefficient.

Alkylation↗
PubMed1984

Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry.

We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational correlation times of TNS bound to apomyoglobin are consistent with those expected for this probe rigidly bound to the protein. TNS in propylene glycol also displayed a single exponential decay of anisotropy. These results, in conjunction with the previous successful resolution of multiexponential decays of fluorescence intensity (Lakowicz, J. R., Gratton, E., Laczko, G., Cherek, H., and Limkeman, M. (1984) Biophys. J., in press; Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J., in press) demonstrate that frequency-domain measurements provide information which is, at a minimum, equivalent to that obtainable from time-domain measurements.

Animals↗
PubMed1984

Structural fluctuations in aspartate transcarbamylase. Succinimide quenching and fluorescence depolarization of tryptophan and tyrosine residues.

The effects of binding of various effector ligands on the dynamics of aspartate transcarbamylase (ATCase, c6r6) and on its regulatory (r2) and catalytic (c3) subunits were characterized by examining succinimide quenching of the intrinsic fluorescence, and by measurement of the lifetime-resolved anisotropies. The lifetimes of the tryptophan residues in c3 and c6r6 are about 1.7 ns while those of tyrosine residues in r2 are 2.7 ns. These lifetimes are not significantly altered by the binding of various substrates, substrate analogs and nucleotides. The effects of ligand binding on the accessibility of both tyrosine and tryptophan residues to the quencher are modest in all cases, though the changes are in the same direction as seen using other physicochemical techniques such as hydrogen exchange (M. Lennick and N.M. Allewell, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 6759). The tryptophan residues in both c3 and c6r6 are immobilized whereas the tyrosine residues of r2 have some motional freedom. Ligands have no effect on the immobilized tryptophan residues in c3 and c6r6, while binding of nucleotides to r2 results in a small decrease in the motional freedom of the tyrosine residues. These results suggest that the protein matrix around the aromatic amino acids in r2, c3 and c6r6 is rather rigid and that local effects of ligands on the dynamics of these residues, and that of the surrounding protein matrix, are minor. They are in general agreement with the results of the crystal structure determination (R.B. Honzatko et al., J. Mol. Biol. 160 (1982) 219).

Aspartate Carbamoyltransferase↗
PubMed1984

Resolution of mixtures of fluorophores using variable-frequency phase and modulation data.

We measured fluorescence phase shift and modulation data for one-, two- and, three-component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using the least-squares procedure described in the preceding paper (Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463-477). Using data obtained at a single emission bandpass, the lifetimes and preexponential factors of two-component mixtures could be easily resolved if the lifetimes differed by a factor of 2. With currently available instrumental stability, three-component mixtures could be resolved when the overall range of decay times was 10-fold, (e.g., 1.3, 4.4, and 12 ns). Measurement of phase and modulation data at several emission wavelengths, where the ratio of the preexponential factors varied, enhanced our ability to resolve closely spaced two and three-component decays. Two-component mixtures could then be resolved if the lifetimes differed by 30% (4.4 and 6.2 ns). Also, the multiple-wavelength data allowed the lifetimes and emission spectra of the three-components of a mixture to be resolved. These results demonstrated that resolution of multiexponential decay laws was possible using frequency-domain phase-modulation fluorometry.

Anthracenes↗
PubMed1983

Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins.

We examined the dynamics of oxygen quenching and depolarization of tyrosine fluorescence in small peptides and proteins lacking tryptophan. The oxygen-quenching constants and the apparent correlation times for fluorescence depolarization were found to be sensitive to the conformational state of the proteins. For small peptides and random coil proteins, the oxygen bimolecular quenching constants indicated complete accessibility of the tyrosine residues to collisions with oxygen. For folded proteins, the quenching constants were about 2-fold smaller, indicating only limited shielding of the tyrosine residues from oxygen by the protein matrix. We also used the steady state anisotropies, measured under conditions of oxygen quenching, to estimate the motional freedom of the tyrosine residues. For random coil proteins, such as a tyrosine copolymer and histones at low pH, the data clearly indicated that depolarization occurs due to subnanosecond segmental motions of the tyrosine residues which are independent of overall protein rotation. For some folded proteins, including bovine pancreatic trypsin inhibitor, the data are consistent with, but do not unambiguously demonstrate, motional freedom of the residues. In these cases, energy transfer among tyrosine residues may also contribute to the observed depolarization. Overall, these results indicate that the rate and extent of tyrosine rotation in proteins depend upon the conformation of the protein and the specific protein under observation.

Chemical Phenomena↗
PubMed1983

Rotational freedom of tryptophan residues in proteins and peptides.

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.

Kinetics↗
PubMed1982

In vitro uptake and transfer of chlorinated hydrocarbons among human lipoproteins.

The uptake, distribution, and exchange of chlorinated hydrocarbon insecticides (dieldrin and chlordecone) and biphenyls (2,4,5-2',4',5'-hexachlorobiphenyl and 3-chlorobiphenyl) among human lipoproteins was examined by fluorescence quenching, gel filtration, and ultrafiltration. The chlorinated hydrocarbons were rapidly taken up from solution or silica particles by lipoproteins. The distribution of chlorinated hydrocarbons among the lipoproteins was independent of the amount taken up by the lipoproteins. The partition coefficient for each lipoprotein and the serum concentration of individual lipoproteins determined the distribution pattern of chlorinated hydrocarbons among lipoproteins. The chlorinated hydrocarbons attached to albumin or one of the lipoproteins were rapidly transferred to all other lipoproteins. The exchange was complete in less than one minute. The role of rapid exchange of chlorinated hydrocarbons among lipoproteins in removal of these chemicals from blood and distribution to other tissues is discussed.

Biphenyl Compounds↗
PubMed1981

Interaction of insecticides with human plasma lipoproteins.

The binding of chlorinated hydrocarbon, carbamate and organophosphate insecticides to human low density plasma lipoproteins (LDL) and high density plasma lipoproteins (HDL) was studied at pH 7.0 and 16 degrees C and 26 degrees C by equilibrium dialysis, difference spectra and fluorescence. The results suggest interaction to be a partitioning rather than a stoichiometric binding process. Distribution is related to lipid content and composition of the lipoproteins. The K-values vary from 3 x 10(5)M-1 for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) to less than 10 M-1 for nicotine and aldicarb, and delta G degrees tr is in the range of 7400 cal for DDT to less than 1000 cal for aldicarb and nicotine. The K and delta G degrees tr are inversely related to the water solubility of the insecticides. A significant role of plasma lipoproteins in the transport of slightly water soluble insecticides is suggested.

Humans↗
PubMedDate not supplied

Transport of conjugates of toxicants by blood proteins.

Binding of naphthol and its glucoside and glucuronide conjugates by blood proteins was studied in vitro and in vivo. Binding was found to be primarily to the albumin fraction of human blood and the binding constants were moderate to low. Both in vivo (mice) and in vitro (human) experiments suggest that a substantial portion of naphthol and two conjugates are transported in bound form to the site of elimination.

Animals↗