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J M Vanderkooi

Publications and source records attributed to J M Vanderkooi.

At least 19 recordsLinked to original sources
PubMed1992

Vibronic energy map and excited state vibrational characteristics of magnesium myoglobin determined by energy-selective fluorescence.

The vibrational frequencies of the singlet excited state of Mg-substituted myoglobin and relative absorption probabilities were determined by fluorescence line-narrowing spectroscopy. These spectra contain information on the structure of the excited state species, and the availability of vibrationally resolved spectra from excited state biomolecules should aid in elucidating their structure and reactivity.

Animals↗
PubMed1992

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Dioxide↗
PubMed1992

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.

Alcohol Dehydrogenase↗
PubMed1992

More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study.

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.

Biophysical Phenomena↗
PubMed1991

Intrinsic tryptophan phosphorescence as a marker of conformation and oxygen diffusion in purified cytochrome oxidase.

Cytochrome oxidase exhibits phosphorescence from tryptophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween-20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern-Volmer quenching constant of approximately 5 x 10(7) M-1.s-1 at 5 degrees C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from tryptophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen.

Animals↗
PubMed1991

Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.

Horseradish Peroxidase↗
PubMed1991

Oxygen in mammalian tissue: methods of measurement and affinities of various reactions.

Oxygen is the primary oxidant in energy-producing biological reactions and is also involved in the synthesis and degradation of many structural and regulatory molecules of physiological importance. This review discusses the advantages and limitations of the currently available methods for measuring oxygen in mammalian tissue and bodily fluids. These include 1) the effects of O2 on the relaxation time of molecules excited by electromagnetic radiation and observed by optical (fluorescence and phosphorescence) and magnetic (nuclear magnetic resonance and electron paramagnetic resonance) techniques, 2) the polarographic and galvanic reduction of oxygen at metal surfaces, 3) in vivo spectrophotometry of intrinsic redox systems, 4) manometry and tonometry, and 5) mass spectroscopy. The values of tissue oxygenation obtained with these techniques are compared with the Michaelis constant values for oxygen of almost 60 oxygen-consuming enzymes involved in mammalian tissue metabolism.

Animals↗
PubMed1990

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes.

Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depends upon the charge distribution of side groups on the porphyrins. Probes that partition into the membrane are sensitive to phase transitions in lecithin artificial membranes. In the mitochondria these membrane probes are within transfer distance from tryptophans in membrane proteins. Although absolute concentrations of oxygen in membranes cannot be determined by this method, by comparing the oxygen dependence of a probe in the aqueous phase with that in the membrane phase under actively respiring and inhibited conditions, it is possible to examine the extent of O2 depletion at the mitochondrial surface. We show that at oxygen tensions of 0.2 microM and higher these gradients are insignificant at usual oxygen consumption rates of mitochondria.

Animals↗
PubMed1990

Reactions of excited triplet states of metal substituted myoglobin with dioxygen and quinone.

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.

Benzoquinones↗
PubMed1990

Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence.

Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface--i.e., kq = Aexp(-r/rho), where A contains a geometrical factor dependent on tryptophan burial and surface geometry [corrected]. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electron-transfer rate on distance in a protein environment and evaluate the distance parameter, rho, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donor-acceptor pair with ket = koexp(-r/rho), ko approximately 10(9) sec-1.

Enzymes↗
PubMed1990

The anaerobic photolysis of lens alpha-crystallin: evidence for triplet state mediated photodamage.

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.

Anaerobiosis↗
PubMed1989

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing.

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.

Energy Transfer↗
PubMed1989

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy.

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗
PubMed1989

Phosphorescence measurements of calf gamma-II, III, and IV crystallins at 77 and 293 K.

Structural and dynamical features of bovine gamma-crystallin tryptophan residues were investigated by phosphorescence measurements at 77 and 293 K. The low temperature phosphorescence spectra and lifetimes of calf gamma-II, III, and IV crystallins did not reflect heterogeneity among the gamma-crystallins. The 0-0 bands were all at 414 +/- 1 nm and the emission lifetimes were all single-exponential with lifetimes of 5.1, 5.3 and 5.3 +/- 0.3 sec, respectively. In contrast, phosphorescence measurements at room temperature were sensitive to subtle differences in exposure, accessibility, and flexibility of gamma-crystallin tryptophan residues. Thorough deoxygenation allowed for measurement of the normally-quenched room-temperature phosphorescence, and we report the first native phosphorescence measurements of lens crystallins at ambient temperature. The emission maxima for gamma-II, III and IV were 446, 442, and 440 +/- 2 nm, respectively. The intensity decay curves were all non-single exponential, and the decays were fit to a sum of two exponentials with lifetimes of 9.1 and 93 msec (gamma-II), 11 and 75 msec (gamma-III), and 4.2 and 68 msec (gamma-IV), +/- 10%. The components of the gamma-II emission were assigned to the four tryptophans based on X-ray structural information. Quantum yields of the phosphorescence emission were in the ratio of 20:7:1 for gamma-II, III and IV, and comparison of lifetimes and quantum yields suggests that tryptophan rigidity increases in the order gamma-IV less than III less than II. Acrylamide quenching constants for the long-lived components of gamma-II and III were roughly equal, while the short-lived tryptophans of gamma-III were an order of magnitude more accessible than those of gamma-II. The wide range of phosphorescence lifetimes and quenching constants allowed for discrimination of distinct contributions to the phosphorescence emission, and we suggest that room-temperature phosphorescence measurements will be an effective tool for studying conformational changes of lens crystallins.

Animals↗
PubMed1989

Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics.

Fluorescence and phosphorescence resemble each other and in many ways can give the same type of information. Both originate from a dipolar interaction between light and the molecule. In this regard, both are polarized and subject to the same type of quenching phenomena. In other respects the information which they divulge are complementary. The fluorescence quantum yield is higher for exposed tryptophans and this is expressed in longer lifetime (Grinvald and Steinberg, 1976); in contrast long lifetime of phosphorescence appears to correlate with burial. Phosphorescence, spin-disallowed, is much longer lived than fluorescence. This allows the structural/dynamic characterization of proteins to be studied on a new time regime. A really remarkable finding of studies of protein phosphorescence is that there is such variability both in phosphorescence lifetime and quenchability. We would interpret this to indicate that the tryptophan environment can range from essentially a crystal, almost comparable in rigidity as found at 77 K, to tryptophans in a flexible environment, almost as flexible as free in solution. An interesting task will be to examine the relationship between the yield and lifetime of phosphorescence and details of the tryptophan environment in terms of rigidity and adjacent amino acids among the proteins with known three dimensional structure.

Luminescence↗
PubMed1988

Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum.

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.

Adenosine Triphosphate↗