Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.     

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.     

Nanosecond fluorescence studies of noncovalent interaction of monomeric and dimeric intercalators with DNA

The noncovalent interaction of 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) and its derivatives, which are potent mutagens isolated from L-glutamic acid pyrolysate, with calf thymus DNA was studied by steady-state and nanosecond fluorescence spectroscopies. The fluorescence of these compounds exhibits static quenching by noncovalent interaction with DNA. Fluorescence lifetimes of the free and intercalated states of these compounds were determined to be 9-10 and 0.5-1 ns, respectively. The bisintercalative effect of the dimeric analogue of Glu-P-2, bis(Glu-P-2)spermine (2GP-SP), to DNA was also investigated. This 2GP-SP, which has two Glu-P-2 moieties at each end of spermine, indicates a strong intramolecular interaction exhibiting remarkable quenching of fluorescence spectrum and lifetime (tau = 3.5 ns) in the absence of DNA. In the presence of DNA, however, the 3.5-ns lifetime component of fluorescence disappeared, and a two-exponential decay of fluorescence (t = approximately 10 and 1.5 ns) was observed at a DNA concentration of more than approximately 0.001 mM P, while the solution containing a very dilute DNA concentration (less than or equal to 0.001 mM P) exhibits a three-component decay of fluorescence (1.5, 3.5, and approximately 10 ns). The potent bis intercalation of two moieties in 2GP-SP with an identical DNA molecule was suggested by the DNA-concentration dependence of these fluorescence lifetimes and their intensity.     

Phosphagen kinase evolution. Expression in echinoderms

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140-155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.     

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.     

Distributions of fluorescence decay times for parinaric acids in phospholipid membranes

Analysis of fluorescence decay data for probes incorporated into model or biological membranes invariably requires fitting to more than one decay time even though the same probe exhibits nearly single-exponential decay in solution. The parinaric acids (cis and trans) are examples of this. Data are presented for both parinaric acid isomers in dimyristoylphosphatidylcholine membranes collected to higher precision than normally encountered, and the fluorescence decays are shown to be best described by a smooth distribution of decay times rather than by a few discrete lifetimes. The temperature dependence of the fluorescence decay reveals a clear shift in the distribution to longer lifetimes associated with the membrane phase transition at 23.5 degrees C. The physical significance is that fluorescence lifetime measurements appear to reflect a physical process with a distribution of lifetimes rather than several distinct physical processes.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.     

Proteolytic susceptibility of creatine kinase isozymes and arginine kinase

The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.     

Frequency domain measurements of the fluorescence lifetime of ribonuclease T1.

Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report.     

Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.     

The dead-end elimination method,tryptophan rotamers,and fluorescence lifetimes

The Dead-End Elimination method was used to identify 40 low energy microconformations of 16 tryptophan residues in eight proteins. Single Trp-mutants of these proteins all show a double- or triple-exponential fluorescence decay. For ten of these lifetimes the corresponding rotameric state could be identified by comparing the bimolecular acrylamide quenching constant (k(q)) and the relative solvent exposure of the side chain in that microstate. In the absence of any identifiable quencher, the origin of the lifetime heterogeneity is interpreted in terms of the electron transfer process from the indole C epsilon 3 atom to the carbonyl carbon of the peptide bond. Therefore it is expected that a shorter [C epsilon 3-C[double bond]O] distance leads to a shorter lifetime as observed for these ten rotamers. Applying the same rule to the other 30 lifetimes, a link with their corresponding rotameric state could also be made. In agreement with the theory of Marcus and Sutin, the nonradiative rate constant shows an exponential relationship with the [C epsilon 3-C[double bond]O] distance for the 40 datapoints.     

A physicochemical comparison of the isozymes of creatine kinase from rabbit brain and muscle

A comparison of specific structural features of creatine kinase from rabbit muscle and brain was undertaken to determine if the observed isozyme specific differences in catalytic cooperativity are related to conformational differences, particularly differences in packing density. The intrinsic fluorescence of the brain isozyme is 2-fold higher than the muscle isozyme. In the denatured state, both proteins display the characteristic red shift in emission maximum; however, the emission intensity of the brain isozyme increases only 5% upon denaturation compared to nearly 100% increase for the muscle protein. The fluorescence lifetimes are 2.65 ns (67%) and 0.48 ns for native muscle enzyme and 4.38 ns (65%) and 0.80 ns for brain enzyme. Upon denaturation, the lifetimes are 3.98 ns (77%) and 0.99 ns for muscle protein and 3.82 ns (79%) and 0.86 ns for brain protein. Stern-Volmer plots of quenching by acrylamide are essentially the same for both native isozymes indicating that the differences of the intrinsic fluorescence of the native proteins are not due to differences in solvent accessibility. The spectral and lifetime differences in the isozymes in the native state and changes accompanying denaturation are consistent with the occurrence of energy transfer in native muscle isozyme. The rotational correlation times of 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonate conjugated proteins, derivatized at the active site reactive thiol, are best described by two term decay laws. The slower rotations, 45.1 ns (75%) and 40.6 ns (71%) reflect overall macromolecular rotation for the muscle and brain isozymes, respectively. The faster motions, 2.4 ns for muscle isozyme and 0.4 ns for the brain isozyme, are attributed to the probe or probe associated segmental motions and indicate these motions are more restricted in the muscle protein. Reactivity of creatine kinase (2.5-10 microM) with the amino-specific reagent trinitrobenzene sulfonate (0.4-2 mM) was analyzed by pseudo-first-order and second order models, neither of which was adequate for the entire range of data. However, in every case, the rate constants were faster for brain creatine kinase but the extent of reaction was greater for muscle creatine kinase. The faster initial reactivity of the brain isozyme is consistent with greater accessibility for lysine derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intramembranal events in the biosynthesis of peptidoglycan in Gaffkya homari

In order to monitor the intermediates involved in nascent peptidoglycan (PG) assembly in Gaffkya homari, a pulse/chase assay utilizing UDP-MurNAc-Ala-DGlu-Lys(N epsilon-Dns)-DAla-DAla [Dns (dansyl) = 5-(dimethylamino)naphthalene-1-sulfonyl] was devised. The perturbation introduced by the dansyl group provided a means for separating the synthesis of nascent PG into discrete stages. Together with paramagnetic quenching of the fluorophore by n-doxylstearic acids (n = 5, 7, 12, 16; doxyl = N-oxy-4',4'-dimethyloxazolidine), this assay allows one to observe the synthesis of undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc and its utilization for the formation of dansyl-labeled PG by fluorescence emission and by change in specific positional quenching. The utilization of the dansylated lipid disaccharide-pentapeptide occurs without a lag, whereas the formation of the chromatographically immobile dansylated PG occurs with a lag of 4-6 min. Membrane-associated undecaprenyl diphosphate-MurNAc-(N epsilon-Dns)-pentapeptide was quenched primarily by 7-doxylstearate. In contrast, the fluorophore of the undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc was quenched primarily by 5-doxyl- and 16-doxylstearates. In the chase phase of the assay, quenching by 16-doxylstearate decreased at a faster rate than that by 5-doxylstearate during the formation of dansyl-labeled PG.     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase.

The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.     

Effects of anions on a monomeric and a dimeric arginine kinase.

1. Some effects of anions on the rates of phosphoarginine synthesis by monomeric (lobster) and by dimeric (Holothuria forskali) arginine kinases are reported. 2. As with creatine kinase, acetate ions activate both enzymes: Cl- was also found to activate both although this was an inhibitor of creatine kinase. 3. NO3- inhibits the lobster enzyme. Inhibition is of the mixed type with respect to MgATP. Ki greater than Ki' and Ks greater than Ks' indicating that the presence of NO3- promotes the binding of substrate and vice versa. 4. NO3- alone has no effect on the difference spectrum of the lobster enzyme but in the presence of arginine, MgATP, MgADP, MgAMP or MgIDP the difference spectrum is greatly enhanced. A profound effect on the ionization state of tyrosine residues is inferred. 5. With the Holothuria enzyme low concentrations of NO3- activate in a manner that is competitive with arginine. Higher concentrations cause inhibition of the mixed type with respect to arginine in a similar manner to that found with MgATP for the lobster kinase. 6. Of a range of anions tested only NO3- and NO2- enhanced the inhibition of enzyme activity by MgADP, indicating the formation of a pseudo-transition-state dead-end complex, enzyme-arginine-NO3--MgADP. The effect was essentially independent of temperature with the Holothuria enzyme, but with the lobster enzyme was much less marked and temperature dependent. The difference may reflect the different stabilities of the monomer and dimer enzymes, although with neither arginine kinase is the stabilization of the dead-end complex as marked as is found with creatinine kinase.     

Synthesis and characterization of the dansyltyrosine derivatives of porcine pancreatic colipase

Steady-state and time-resolved fluorescence techniques were used to study dansyltyrosine derivatives of porcine pancreatic colipase. Nitration, reduction, acylation, and dansylation reactions were utilized to synthesize two fluorescently labeled colipases: (o-aminodansyltyrosine 55 porcine colipase) (DNStyr55PC) and o-aminodansyltyrosine 59 porcine colipase (DNStyr59PC). DNStyr55PC was 200% active, while the DNStyr59 derivative maintained 80% activity in a pH stat assay. Emission spectra, lifetime analysis, acrylamide quenching, polarization, and anisotropy decay studies indicated that Tyr55 was located on the solvent-exposed surface of the protein, where the fluorophore experienced free rotation. Identical experiments done on DNStyr59PC indicated that Tyr59 was in a partially buried environment and the motion of the dansyl tyrosine group was hindered. The double-exponential decay of the fluorescence emission of N-acetyl-o-aminodansyltyrosine ethyl ester (DNStyr) and the DNStyr derivatives of colipase was investigated with pH, temperature, solvent, and emission-resolved-lifetime experiments. The existence of excited-state processes was eliminated in both pH and emission-resolved-lifetime experiments, whereas temperature studies indicated either a rotational isomer or a differential solvent quenching mechanism for multiple decay kinetics. These experiments also showed that DNStyr was a sensitive probe of solvent polarity and viscosity, but not of pH.