Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

Second-derivative spectroscopy of proteins: Studies on tyrosyl residues

Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.     

Long-wavelength chlorophyll forms in Photosystem I from pea thylakoids

Absorption maximum positions of three LW Chl forms in pea chloroplasts were estimated using 77 K excitation spectra of fluorescence detected in their maxima (720, 732 and 746 nm). The 705, 714 and 723 nm components were revealed in the second derivative plots of the excitation spectra. The same maxima were found in normalized excitation spectra obtained with dividing excitation spectra by absorption spectrum. It was confirmed that the observed maxima belong to absorption of LW fluorescing Chl forms. The same maxima were displayed in an action spectrum of P700 oxidation measured at room temperature. It confirms the energy transfer from LW Chl forms to P700. Close to 50% efficiency of bulk Chl forms in both excitation of LW fluorescence and P700 oxidation was found. Analysis of the shape of normalized excitation spectra suggests that there is no energy exchange among LW Chl forms. Their location and physiological role are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Absorption and fluorescence spectra and molecular organization of bacteriochlorophyll in reaction centers of Rhodopseudomonas spheroides

Second derivative spectroscopy, computer curve analysis and Stepanov's equation show that the absorbance and fluorescence spectra of primary electron donor in reaction center of Rhodopseudomonas sphaeroides are splitting each into two asymmetric Gaussian components. Their absorption maxima at -196 degrees are 880 and 896 nm and emission maxima-906 and 923 nm, respectively. The absorption spectrum of Bchl-800 splits in the near infrared region into two bands with maxima at 790 and 803 nm. These components are ascribed to an exciton coupling in the two dimers of bacteriochlorophyll in the reaction center. The Qy transition moments of the two bacteriochlorophyll molecules of primary electron donor make an angle of 110 degrees and the angle between two Qy transitions of the pigment in Bchl-800 dimer is 150 degrees. The distance between the centers of chromophores in the dimers is estimated to be 8-11 A.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

Circular dichroism of rhodopsins and porphyropsins

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 321–700 nm: an α peak at about 520 nm and a small β peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the α peaks of the porphyropsins are larger than the α peaks of the rhodopsins, while the β peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blue-shifted from the corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250–320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.     

Analysis of the ESR spectrum of synthetic dopa melanin

The 35 GHz ESR spectrum of frozen aqueous suspensions of synthetic melanin from autoxidation of dopa is asymmetric in the pH range 3-12. This asymmetry increases with increasing pH. A detailed computer analysis of second-derivative spectra suggests that the asymmetry is a result of two factors: approximately axial anisotropy of the g-tensor of the radical species; superposition of ESR spectra arising from a total of four radical species. The relative amounts of these individual spectra vary in a pH-dependent manner. Anisotropy of spectra varies with pH, probably due to pH-induced changes in the delocalization of the unpaired electrons. Thus the species present at high pH are suggested to be relatively localized radical anions, while the species detected at low pH are suggested to be protonated forms of the high pH species in which the unpaired electron is more extensively delocalized.     

Conformational changes of hemoproteins as evidenced by second derivative spectroscopy of aromatic amino acids

Based on a comparison of the 2nd derivative spectra of cytochrome P-450 LM2, cytochrome c and hemoglobin with the corresponding mixtures of aromatic amino acids and the individual aromatic amino acids a significant red shift of the maxima of the absorption bands appearing as distinct minima in the 2nd derivative spectrum have been observed in the proteins. This red shift has been shown to be caused by a lowered polarity of the immediate surroundings of the chromophores. The band positions of the aromatic amino acids in the 3 investigated hemoproteins are nearly the same and therefore do not allow to qantitate conformation dependent spectral changes of the chromophores. In agreement with X-ray crystallographic data aromatic amino acids of cytochrome c and of hemoglobin are subjected to spectral changes at the transition from the oxidized to the reduced state. These redox linked conformational changes are indicated by significant changes of the amplitudes of the aromatic acids in the 2nd derivative spectrum. A quantitative evaluation of the amplitudes revealed characteristics specific for cytochrome c and hemoglobin. Surprisingly the 2nd derivative spectrum in the median UV-region of cytochrome P-450 LM2 does not show any significant change at reduction.     

The individual tyrosines of proteins: their spectra may or may not differ from those in water or other solvents

The overall derivative spectrum of a protein is the sum of the individual derivative spectra just as the overall ultraviolet spectrum of a protein is the sum of its component parts. The RNase and DNA binding protein Sso7d has two tyrosines and one tryptophan. We used two mutant forms of the protein to show that the individual aromatics contribute derivative spectra that can be explained on the basis of their environments. We used mutant forms of iso-1-cytochrome c to estimate the contributions of the single tryptophan and three of the five tyrosines to the overall derivative spectrum. The tryptophan spectrum is not exceptional. The comparable tyrosine spectra are more complex. The derivative spectrum of individual tyrosines does not correspond to that expected on the basis of concentration. This is a reflection of two factors: (1) the extent to which mutations are sensed distally through the introduction and compression of packing defects; and (2) the extent to which electronic transitions of tyrosine are influenced by nearby atoms. This influence could take the form of tyrosine residing in an area where the dielectric coefficient is not uniform; it could also result from tyrosine bumping into neighboring atoms with lower frequency than it does in solution.     

A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures

A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735?nm, and a 2-band spectrum with maxima at 685 and 740?nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.     

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.     

Electric field effects on the chlorophylls,pheophytins, and beta-carotenes in the reaction center of photosystem II

We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.     

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.     

Determination of compound aminopyrine phenacetin tablets by using artificial neural networks combined with principal components analysis

A method for simultaneous, nondestructive analysis of aminopyrine and phenacetin in compound aminopyrine phenacetin tablets with different concentrations has been developed by principal component artificial neural networks (PC-ANNs) on near-infrared (NIR) spectroscopy. In PC-ANN models, the spectral data were initially analyzed by principal component analysis. Then the scores of the principal components were chosen as input nodes for the input layer instead of the spectral data. The artificial neural network models using the spectral data as input nodes were also established and compared with the PC-ANN models. Four different preprocessing methods (first-derivative, second-derivative, standard normal variate (SNV), and multiplicative scatter correction) were applied to three sets of NIR spectra of compound aminopyrine phenacetin tablets. The PC-ANNs approach with SNV preprocessing spectra was found to provide the best results. The degree of approximation was performed as the selective criterion of the optimum network parameters.     

Spectral sensitivity and Color Vision in the Bottlenose Dolphin ( Tursiops Truncatus )

Spectral sensitivity was measured in air in a bottlenose dolphin using a behavioral training technique. The spectral sensitivity curve shows two maxima in sensitivity, one in the near ultraviolet part of the spectrum and the other one in the bluegreen part at about 490 nm. Two wavelength discrimination tasks showed that the dolphin could discriminate two wavelengths from the peak regions of the two maxima of the spectral sensitivity function, but not between two wavelengths lying within the broad maximum of the curve in the bluegreen part of the spectrum. Possible underlying mechanisms for the shape of the function are discussed.     

Formation of a bis(histidyl) heme iron complex in manganese peroxidase at high pH and restoration of the native enzyme structure by calcium

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high- to low-spin alkaline transition occurs at approximately 2 pH units lower in an F190I mutant MnP when compared to the wild-type enzyme. Herein, we provide evidence that these spectral changes are attributable to the formation of a bis(histidyl) heme iron complex in both proteins at high pH. The resonance Raman (RR) spectra of both ferric proteins at high pH are similar, indicating similar heme environments in both proteins, and resemble that of ferric cytochrome b(558), a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visible spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 nm, resembling the absorption spectrum of ferrous cytochrome b(558). RR spectra of the reduced wild-type and F190I mutant proteins at high pH are also similar to the RR spectrum of ferrous cytochrome b(558), further suggesting that the alkaline low-spin species is a bis(histidyl) heme derivative. No shift in the low-frequency RR bands was observed in 75% (18)O-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that addition of Ca(2+) to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn(2+), Mg(2+), Zn(2+), or Cd(2+), do not restore the enzyme under the conditions studied.     

FTIR spectroscopic analyses of human placental membranes

We investigated the differences in the Fourier transform infrared (FTIR) spectra of normal and abnormal human placentas. Normal placentas, placentas with infant intrauterine growth restriction (IUGR), and placentas from mothers with diabetes mellitus (DM) were used, none of which had been treated before measurement. The tissues were divided into three parts: the upper one-third portion (P1), the middle portion (P2), and the lower one-third portion (P3). Placental tissues were also investigated histochemically. The differences of the main second-derivative FTIR spectra among P1, P2, and P3 in normal placentas were observed in bands appearing between 1080 and 1090 cm(-1). Bands in P2 were observed at 1083 cm(-1), which was significantly higher than that in P3 (p < 0.05). The spectrum of P2 tissue in placentas with infant IUGR had a peak at 1081 cm(-1), which was significantly different from those of P1 and P3 (p < 0.05). In placentas with DM, the P2 band was shifted to a peak at 1088 cm(-1). These data were well correlated with the histochemical sugar-chain staining pattern of the P2 portion of the placenta. Our data suggested that this IR technique is applicable to the clinical diagnosis of diseases in the gynecological field.     

Spectral sensitivity and Color Vision in the Bottlenose Dolphin (Tursiops Truncatus)

Spectral sensitivity was measured in air in a bottlenose dolphin using a behavioral training technique. The spectral sensitivity curve shows two maxima in sensitivity, one in the near ultraviolet part of the spectrum and the other one in the bluegreen part at about 490 nm. Two wavelength discrimination tasks showed that the dolphin could discriminate two wavelengths from the peak regions of the two maxima of the spectral sensitivity function, but not between two wavelengths lying within the broad maximum of the curve in the bluegreen part of the spectrum. Possible underlying mechanisms for the shape of the function are discussed.     

Resolution of Fluorescence Emission Spectra with Various Lifetime Components of D1/D2/Cyt b-559

PS Ⅱ reaction center D1/D2/Cyt b-559 purified from chloroplasts of spinach has four components of fluorescence decaying with lifetimes of 1.0 ns, 5.9 ns,24 ns,and 73 ns whose fractions to total fluorescence yield are 0. 05,0.34,0. 35 and 0.26 respectively. The fluorescence emission spectra of these lifetime components are closely overlapping, and only one peak is shown in steady state emission spectrum. Based on the hardware analysis of phase fluorometry,by selection of the detector phase angle,the emission from various components could be individually suppressed. If the 5.9 ns component was suppressed, the emission spectrum was red-shifted. On the contrary, the emission spectrum was blue-shifted when 73 ns component was suppressed. Based on the software analysis, the individual emission spectra were resolved with three lifetime components by measuring phase and modulation data at various wavelength. Compared with steady state spectrum,the emission maximum wavelength of 5.9 ns component was blue-shifted from 68nm to 680 nm,but those of 24 ns and 73 ns components were red-shifted to 685 nm and 683 nm respectively.     

Clean absorption mode NMR data acquisition based on time-proportional phase incrementation

Clean absorption mode NMR data acquisition is presented based on mirrored time domain sampling and widely used time-proportional phase incrementation (TPPI) for quadrature detection. The resulting NMR spectra are devoid of dispersive frequency domain peak components. Those peak components exacerbate peak identification and shift peak maxima, and thus impede automated spectral analysis. The new approach is also of unique value for obtaining clean absorption mode reduced-dimensionality projection NMR spectra, which can rapidly provide high-dimensional spectral information for high-throughput NMR structure determination.