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Objectives Thromboembolic complications are present in 0.8%–16.8% of the cases after radical prostatectomy (RP). Association between elevated plasma von Willebrand factor (VWF) levels—as an endothelial activation marker—and increased risk of thrombotic events has been evidenced. We aimed to elicit new data on the VWF after RP in prostate cancer patients and explore the role of it as a thrombotic risk factor. Upon perioperative plasma VWF levels (VWF:Ag) its collagen-binding (CB) activity (VWF:CB), multimerization, and cleaving enzyme (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type repeats, motif 1, type 13]) of the VWF multimers were quantitated along with Factor VIII and routine laboratory parameters in this observational pilot study. Methods Plasma samples of 24 prostate cancer patients were collected before (-1 day; D-1) and after RP (1 hour, 6 days, 1 month, and 10 months; H1, D6, M1, and M10). VWF:Ag, VWF:CB, ADAMTS13:Ag were measured by ELISA, and the multimer distribution by electrophoresis and quantitative densitometry. Factor VIII, fibrinogen, D-dimer, and other routine laboratory parameters were determined as well. Preoperative values served as baselines which were compared to controls (24 healthy individuals). Results VWF:Ag and CB elevated by 122% and 143% respectively at H1 after RP then plateaued at D6 compared to baseline values. ADAMTS13/VWF:Ag ratio reduced by 41% at H1, and by 46% at D6, meanwhile the ratio of high molecular weight multimers increased as well. Values returned to baseline at M1 and further reduced to the levels of the controls at M10. All of the 24 patients at H1 and D6 and 14 at M10 were in potential prothombotic state according ROC analysis of the VWF parameters as indicators. Conclusions Prostate malignancy and then surgical stress, and inflammatory reactions induced release of VWF from the endothelial cells, along with an increasing amount of large multimers and relative reduction of ADAMTS13 level. Because these changes mark a prothrombotic state even at M1 after RP, more than 1 month follow-up and prophylactic targeting through the thrombotic and inflammatory activity of the VWF is proposed. Evaluation of VWF parameters provides new information about the long-term disturbances of primary hemostasis after radical pelvic oncologic surgery like RP and might improve the understanding the physiological and pathological recovery.

Background Little is known about the potential role of central obesity among men. Our first aim was to confirm what is already known from prior research, namely that both BMI and WHR are inversely associated with selected semen parameters. Our second aim was to examine the potential role of central obesity by assessing if there was a difference between BMI and WHR regarding their relationships to these selected semen parameters. Methods In this cross-sectional study between January 2011 to January 2018, we analyzed semen samples from 1169 patients who visited an andrology clinic in Budapest for infertility reasons. Variables assessed were: body measurements (height, weight, waist circumference, and hip circumference), and the results of semen analysis (sperm concentration, total sperm count, progressive sperm motility, and normal sperm morphology). Results The mean height and weight were 180.6 cm and 87.3 kg, respectively – the mean BMI was 26.8. The mean waist and hip circumferences were 100.9 cm and 94.8 cm, respectively – the mean waist to hip ratio was 0.94. The mean sperm concentration, total sperm count, and percents of progressive motility and normal morphology were 48.7 M/ml, 165 million, 21.2, and 4.8%, respectively. Both BMI and WHR were significant correlates in all semen parameter regression models. When comparing the parameter estimates for BMI with those for WHR for each semen parameter, the parameter estimate for WHR was significantly lower (indicating a stronger negative association) than that for BMI for progressive motility and total sperm count, but not for normal morphology or concentration. Conclusions Our study is the first to examine, using a large patient sample, the potential role of central obesity by comparing the difference between BMI and WHR as they relate to selected semen parameters. Our findings indicate a potential role of central obesity for progressive motility and total sperm count, but not for normal morphology and concentration. Despite the limitations and the exploratory nature of this study, we can conclude that our results point to a potential role of central obesity in male infertility, but this finding should be confirmed and further explored in future research. Trial registration The trial was retrospectively authorized after the data collection on September 24, 2018. Registration number: SE RKEB: 169/2018.

The Hungarian Case-Control Surveillance of Congenital Abnormalities (HCCSCA) is one of the largest case-control data sets of CA-surveillance in the world. We unified all data collected in the HCCSCA between 1980 and 2009 into a new, validated single database that is now open for examination. The details of this unified database are given in this paper. The total number of cases and control newborns is 32,345 and 57,231, respectively. The overall prevalence of CAs recorded in the HCCSCA was 10.7/1000 live-births. Data available for each pregnancy are: CA(s), gender, birth year/month/date, birth weight, gestational age, area of mother's living, maternal age, paternal age, birth order, mother's and father's qualification, employment status and type of employment, mother's marital status, outcome of previous pregnancies, maternal diseases during pregnancy (according to pregnancy months), drug intake during pregnancy (according to pregnancy months), folic acid and/or pregnancy vitamin supplement intake (according to pregnancy months), mother's smoking habits and alcohol consumption patterns. The most frequent anomalies detected were ventricular septal defect (2864), atrial septal defect (1895), polydactyly (1499), hypospadias (1083), and unilateral cleft lip ± palate (961). According to ICD-10, 701 diseases have been found to affect case mothers during pregnancy. Eight hundred and sixteen drugs were identified that had been taken by mothers during pregnancy. The authors are absolutely open for any scientific cooperation based on this database.

There are increasing demands nowadays on spatial soil information in order to support environmental related and land use management decisions. Physical soil properties, especially particle size distribution play important role in this context. A few of the requirements can be satisfied by the sand-, silt-, and clay content maps compiled according to global standards such as GlobalSoilMap (GSM) or Soil Grids. Soil texture classes (e. g. according to USDA classification) can be derived from these three fraction data, in this way texture map can be compiled based on the proper separate maps. Soil texture class as well as fraction information represent direct input of crop-, meteorological- and hydrological models. The model inputs frequently require maps representing topsoil features, which refer most commonly to 0–30 cm depth. This is covered by three consecutive depth intervals according to standard specifications: 0–5 cm, 5–15 cm, 15–30 cm. Becoming GSM and SoilGrids the most detailed freely available spatial soil data sources, the common model users (e. g. meteorologists, agronomists, or hydrologists) would produce input map from (the thickness-weighted mean of) these three layers. However, if the basic soil data and proper knowledge is obtainable, a soil texture map targeting directly the 0–30 cm layer could be independently compiled. In our work we compared Hungary's soil texture maps compiled using the same reference and auxiliary data and inference methods but for differing layer distribution. We produced the 0–30 cm clay, silt and sand map as well as the maps for the three standard layers (0–5 cm, 5–15 cm, 15–30 cm). Maps of sand-, silt-, and clay content were computed through composite regression kriging applying Additive Log-Ratio (alr) transformation. In addition to the Hungarian Soil Information and Monitoring System as reference soil data, digital elevation model and its derived components, soil physical property maps, remotely sensed images, land use-, geological-, as well as meteorological data were applied as auxiliary variables. We compared the directly compiled and the synthetized clay-, sand content, and texture class maps by different tools. In addition to pairwise comparison of basic statistical features (histograms, scatter plots), we examined the spatial distribution of the differences. We quantified the taxonomical distances of the textural classes, in order to investigate the differences of the map-pairs. We concluded that the directly computed and the synthetized maps show various differences. In the case of clay-, and sand content maps, the map-pairs have to be considered statistically different. On the other hand, the differences of the texture class maps are not significant. However, in all cases, the differences rather concern the extreme ranges and categories. Using of synthetized maps can intensify extremities by error propagation in models and scenarios. Based on our results, we suggest the usage of the directly composed maps.

AimsIn order to improve diagnostics in pleural effusions, additional value of effusion cholesterol, carcinoembryonic antigen (CEA) and syndecan-2 assays to cytology was studied.MethodsBiomarkers were measured in effusion supernatants from 247 patients, of whom 126 had malignant pleural involvement, and their additional diagnostic efficacy to cytology was assessed.ResultsSyndecan-2 measurement, although gave detectable concentrations in all effusions with highest median value in mesotheliomas, was non-discriminative between different pathological conditions. CEA concentrations exceeding 5 ng/mL cut-off point indicated carcinomas, regardless of pleural involvement, which gave a sensitivity of 62% and specificity of 100% for carcinoma. Cholesterol concentration over 1.21 mmol/L cut-off value indicated neoplastic pleural involvement with 99% sensitivity and ‘merely’ 69% specificity, the latter mainly due to raised levels being associated also with benign inflammatory effusions. Combined CEA and cholesterol determinations increased the sensitivity for diagnosing carcinomatosis from 70% with cytology alone to 84% and established the correct diagnosis in 16 of 31 carcinomatosis cases with inconclusive cytology. Cholesterol measurement alone, with elevated level, in combination with absence of substantial number of inflammatory cells in effusion sediment proved to be a magnificent marker for neoplastic pleural involvement with 99% efficacy, and recognised all 36 such cases with inconclusive cytology.ConclusionsSimultaneous measurement of CEA and cholesterol concentrations in effusion, or at least cholesterol alone, in combination with non-inflammatory fluid cytology, provides additional specific information about neoplastic pleural involvement, and can therefore be used as an adjunct to cytology, above all, in inconclusive cases.

OBJECTIVE: Mechanical evaluation of a novel screw position used for repair in a type III distal phalanx fracture model and assessment of solar canal penetration (SCP). STUDY DESIGN: Experimental study. SAMPLE POPULATION: Disarticulated equine hooves (n = 24) and 24 isolated distal phalanges. METHODS: Hooves/distal phalanges cut in a sagittal plane were repaired with 1 of 2 different cortical screw placements in lag fashion. In group 1 (conventional screw placement), the screw was inserted halfway between the proximal border of the solar canal (SC) and the subchondral bone surface on a line parallel to the dorsal cortex, whereas in group 2, the screw was inserted more palmar/plantar, where a perpendicular line drawn from the group 1 position reached the palmar/plantar cortex. Construct strength was evaluated by 3-point bending to failure. SCP was assessed by CT imaging and macroscopically. RESULTS: Screws were significantly longer in group 2 and in forelimbs. Group 2 isolated distal phalanges had a significantly more rigid fixation compared with the conventional screw position (maximum point at failure 31%, bending stiffness 41% higher). Lumen reduction of the SC was observed in 13/52 specimens (all from group 2), of which 9 were forelimbs. CONCLUSIONS: More distal screw positioning compared with the conventionally recommended screw position for internal fixation of type III distal phalangeal fractures allows placement of a longer screw and renders a more rigid fracture fixation. The novel screw position, however, carries a higher risk of SCP

The general question by what mechanism an "effector" molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.

Binding of photosensitizers to target cells is a crucial step during the photodynamic effect. Sensitizer distribution is a good indication of whether the chemical is a good candidate for perturbing cell membrane integrity. Hence, the photophysical properties of porphyrinoid sensitizers in microheterogeneous systems such as liposomes are of outstanding interest. Here we present a site-selective fluorescence study of liposome systems. Monocomponent, small unilamellar vesicles formed of different phosphatidylcholines with incorporated mesoporphyrin were investigated. The size distribution of liposomes was measured by dynamic light scattering after each step of the experiment. On the basis of fluorescence line narrowing spectra of mesoporphyrin, the inhomogeneous distribution function was determined in order to characterize the photosensitizer location. The dual character of the functions revealed two different locations. Decomposition of the inhomogeneous distribution functions into Gaussians and the analysis of the fit results suggest that one of the locations for mesoporphyrin is between the two lipid layers, and the other one is between the hydrocarbon chains of the lipid molecules.

Mechanical evaluation of a novel screw position used for repair in a type III distal phalanx fracture model and assessment of solar canal penetration (SCP).Experimental study.Disarticulated equine hooves (n = 24) and 24 isolated distal phalanges.Hooves/distal phalanges cut in a sagittal plane were repaired with 1 of 2 different cortical screw placements in lag fashion. In group 1 (conventional screw placement), the screw was inserted halfway between the proximal border of the solar canal (SC) and the subchondral bone surface on a line parallel to the dorsal cortex, whereas in group 2, the screw was inserted more palmar/plantar, where a perpendicular line drawn from the group 1 position reached the palmar/plantar cortex. Construct strength was evaluated by 3-point bending to failure. SCP was assessed by CT imaging and macroscopically.Screws were significantly longer in group 2 and in forelimbs. Group 2 isolated distal phalanges had a significantly more rigid fixation compared with the conventional screw position (maximum point at failure 31%, bending stiffness 41% higher). Lumen reduction of the SC was observed in 13/52 specimens (all from group 2), of which 9 were forelimbs.More distal screw positioning compared with the conventionally recommended screw position for internal fixation of type III distal phalangeal fractures allows placement of a longer screw and renders a more rigid fracture fixation. The novel screw position, however, carries a higher risk of SCP.

The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640 nm that is attributed to a charge-transfer (CT) transition between the a 2u HOMO of ﷿ electrons of the porphyrin ring and the dxy/dyz orbital of the ferric ion. Because the water channel extends to the Fe, it seems likely that the CT band will be sensitive to the hydration of the protein. To study this premise, the protein was incorporated into trehalose/sucrose glasses and the hydration of the sugar glasses was varied. Absorption spectra of HRP in sugar glasses and in glycerol/water were taken in the range 10-300 K. The CT absorption band shows vibronic fine structure. The peak positions are the same in hydrated sugar and glycerol/water but the peak positions change in desiccated sugar glass. The data suggest that in hydrated, but not desiccated, sugar glass, water is retained in the heme pocket. Binding of the competitive inhibitor benzohydroxamic acid to the protein increases the CT absorption and resolution. The effect of benzohydroxamic acid on the Fe as calculated using a combination of density functional theory and molecular mechanics is to stabilize the spin state 3 /2 with respect to 5 /2. At low temperature the widths of the lines in the CT band are narrower for the protein in glycerol/water (glass transition at 150 K) than in trehalose/sucrose (glass formation at 65 °C). This indicates that the CT band is inhomogeneously broadened and sensitive to the solvent. The spectral narrowing of the CT absorption occurs as the temperature decreases over the temperature range studied. Water, as indicated by the OH stretch, also shifts in this range. The findings are discussed in terms of how buried water and nearby charges can modulate the activity of the heme.

Fluorescence line narrowing is a sensitive tool to monitor subtle changes in protein conformation. We restudied the consequences of inhomogeneous broadening on the observed spectra. Vibronic absorption bands of systems with weak electron-phonon coupling can be described by a lorentzian function with width of the order of a few centimeters. For an ensemble of molecules with closely spaced electronic transitions, a single laser frequency will excite many molecules through the broad base (“tail”) of the lorentzian function. This, along with excitation of low-frequency modes, contributes to the unresolved background observed in line-narrowed spectra. Examples are shown for Zn cytochrome c, a fluorescent derivative of Fe cytochrome c. Spectra are compared for the protein in two solvents: glycerol/water or trehalose. Two types of cytochrome, from horse and yeast, are also compared.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170–180K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10–300K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16cm−1 upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A0 band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337–355) shows that such a state presents itself also in myoglobin.

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on δ-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population.

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm -1 . Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of320 cm -1 in the Q(0,0) absorption band that is clearly evident at

Six pieces of grafts, 6.5 mm in diameter, 20 mm in length, were taken from each of 170 cadaver hindlimbs, using the cranial surface of the medial femoral trochlea for harvesting. The age of the horses varied between 4 months and 23 years. 30 limbs under the age of 12 years were selected for transplantation. Three of six grafts were transplanted into the medial femoral condyle using different combinations of tunnel depth and dilation. With ageing, a significant decline in transplantability was detected. In general, mosaicplasty cannot be recommended in horses above 11 years. Based on a previous clinical case (Bodo et al., 2000), a good surface alignment was indeed achieved with a combination of graft length drilling and dilation in most cases. However, the occasional entrapment of cartilage debris under the graft prevented perfect alignment in the present cadaver study in 27% of the grafts transplanted in this manner. Since the protrusion of grafts never exceeded 1.5 mm, we conclude that drilling 3–5 mm deeper than graft length with graft length deep dilation can avoid disadvantageous protrusion of the transplanted hyaline cartilage caps, achieving bone decompression at the same time.

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

The binding of Mg-mesoporphyrin and mesoporphyrin to the primary binding site of human serum albumin (HSA) has been studied in the absence and in the presence of 1,2-dimyristoyl-sn-glycero-3- phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPC/DMPG) liposomes. The equilibrium constants of Mg-mesoporphyrin IX (MgMP) and mesoporphyrin IX (MP) for association to HSA were 1.7 · 107 (M−1) and 2.5 · 107 (M−1), respectively. The association constants for binding to the liposomes were: 1.5 · 104 (M−1) for MgMP, and 3.2 · 104 (M−1) for MP. The smaller value for the association constants of the MgMP relative to MP in both processes are interpreted as the effect of an out of plane position of Mg2+ and possible ligand co-ordination. HSA added to the liposomes with incorporated porphyrins results in the redistribution of bound molecules. For the MgMP this distribution can be interpreted by the competition of two independent binding processes, while the binding kinetics of MP significantly deviates from this model. The difference is explained by supposing a specific interaction between HSA and the liposomes in case of the free-base MP.

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634–636, 644 and 655 nm. At room temperature (293 K) the 628 and 634–636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634–636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634–636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Fluorescence line-narrowing spectroscopy was used to evaluate the true 0,0 band shape from the population distribution of the 0,0 spectral band and to determine the excited-state vibrational spectrum of the porphyrin in Mg protoporphyrin substituted myoglobin (Mg-Mb) and protoporphyrin substituted myoglobin (HH-Mb). The population distribution function for Mg-Mb showed a bimodal distribution, with approximately equal contributions for the two components. The 0,0 energies were centered at 16 716 and 16 873 cm -1 and the widths were 106 and 66 cm -1 for the lower and higher energy forms, respectively

Fluctuations in the polypeptide chain lead to disorder in proteins and to a distribution in the parameters that regulate their functions. Using low temperature (to reduce the fluctuations) and narrow-band lasers (to select one substate among the many forms), high-resolution absorption and fluorescence spectra for chromophores in proteins can be obtained. These spectra reveal information on the kind and extent of disorder in proteins and allow for the determination of the vibrational energies of both ground and excited state molecules, true inhomogeneous spectral width, and kinetic studies of individual protein substates.

Fluorescence line narrowing (FLN) spectroscopy was used to study the vibrational features of Zn porphyrins, including Zn octaethylporphine, Zn mesoporphyrin, Zn deuteroporphyrin and Zn coproporphyrin, and the interaction of these porphyrins with their solvent matrices. A number of ground-state vibrational levels were identified for each of the Zn porphyrins. Six of these levels persistent throughout almost all spectra were analyzed in terms ofcontributions of specific internal vibrational modes of the porphyrins. A number of other vibrational levels were identified as characteristic of the peripheral substituents of Zn porphyrin. Phonon wing contribution in the FLN spectra increased with increased polarity of the solvent matrix, and the Stokes shift calculated from the conventional spectra was correlated with the strength of phonon coupling. Specific vibrational modes were found to couple differently with the solvent and to have different phonon wings. The conventional and energy-selected spectra of Zn coproporphyrin in 5 M guanidine HCI suggest that guanidine ligates to the Zn atom.

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.

At the glass transition temperature of proteins, large scale, anharmonic motions of the conformation become activated. It has been shown that in this temperature range the phosphorescence lifetime of Trp residues that is long (several s) at cryogenic temperatures become efficiently quenched. We report the elaboration of a systematic method based on this observation with the purpose to use this effect for characterizing the global conformational dynamics of proteins. In this method, special conditions were determined under which the onset temperature of phosphorescence quenching can be clearly separated from the slaving effect of fluctuations activated in the solvent, and the relevance of this temperature for the global dynamics of the protein has been verified by parallel experiments on human alpha-oxyFe-betaZn-Hemoglobin, w.t. horse heart Myoglobin and Trp mutant forms of a two-domain protein, yeast phosphoglycerate kinase. The method was used to characterize the role of global dynamics in the allosteric response of human hemoglobin to the binding of effectors as Cl, IHP, DPG, BZF. The quenching effect showed a two state behavior with a characteristic onset temperature sensitive to the presence and quality of allosteric effectors. The data points were fit by a simple two state model and the thermodynamic parameters - enthalpy and entropy changes characteristic for the quenching effect were discussed in terms of oxygen hopping models. The study on hemoglobin verifies the concept that in allosteric regulation, the global dynamics of the protein tetramer plays important role.Acknowledgements: the authors thank Prof. T. Yonetani of University of Pennsylvania for kindly providing Zn-PP- substituted human Hb for the studies. J. Fidy thank the Hung. Ministry of Health for support by grant no. ETT 512/2006.

The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands’ anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSSh), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSSh exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSSh has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of n for the protein in aqueous solutions and in trehalose ysucrose glasses. We have previously CO observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol y water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe-CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band. 2003 Elsevier Science B.V. All rights reserved.

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.

Aim—To determine whether assays of carcinoembryonic antigen (CEA) and cholesterol in ascites add diagnostic value to cytology. Methods—The additional diagnostic efficacy of the biochemical assays was studied in the ascitic fluid from 130 patients, of whom 57 had peritoneal carcinomatosis. All diagnoses were verified by subsequent necropsy and/or histology. Results—CEA concentrations over 5 ng/ml indicated carcinomas, occasionally without peritoneal involvement of the tumour. However, increased values were significantly more common in cancer with peritoneal involvement (p < 0.01), giving a sensitivity of 51% and specificity of 97% for carcinomatosis. A cholesterol value exceeding 1.21 mmol/litre was found in 93% of cancers with peritoneal involvement, but it was not entirely specific (96%) for carcinomatosis. Simultaneous increases in CEA and cholesterol concentrations were specific for carcinomatosis and this combination increased the sensitivity for diagnosing carcinomatosis from 77% with cytology alone to 88%. The correct diagnosis could thus be made in five of 12 cases with inconclusive cytology. Conclusions—The measurements of both CEA and cholesterol concentrations in ascites give additional specific information about peritoneal carcinomatosis and can therefore be a useful adjunct to cytology—in particular, in inconclusive cases. Key Words: ascitic fluid • carcinoembryonic antigen • cholesterol • cytology

Fluorescence line narrowing in combination with high pressure was used to obtain information on the Mg-mesoporphyrin molecule incorporated into a protein mimetic matrix (DMSO). Two distinct conformers were found, their population was changed when pressure was applied. ∆V between the conformers, and the excited state vibrational levels of the chormophore, were determined. When the chromophore was embedded into horseradish peroxidase, there was only one dominant conformer in the inhomogeneous distribution function.

Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.

The porphyrin in metal-free cytochrome c molecules was investigated using fluorescence line narrowing spectroscopy. An energy map is defined to illustrate the environmental effects on the vibronic structure of a chromophore. In the approximation of pure electronic distortion, the population distribution in the energy scale can be characterized by a function. An intuitive definition of this distribution function is determined for the porphyrin in cytochrome c, and the validity of this approximation is experimentally verified. In addition, numerous excited state vibrational levels suggest a method to quantify the relative absorption probabilities for (0,0)yields(1,k) transitions.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Fluorescence line narrowing (FLN) spectroscopy was used to study the role of the polypeptide chain in influencing the spectrum of Zn-substituted cytochrome c (Zn cyt c) and metal-free cyt c (porphyrin cyt c). For both derivatives the spectra show characteristics of relaxed fluorescence from an inhomogeneously broadened sample. Zero phonon lines and phonon wings can be clearly distinguished, and vibrational frequencies of the ground and excited states were identified. The inhomogeneous distribution width for porphyrin cyt c is slightly wider than that of Zn cyt c and a second population of molecules was apparent in the porphyrin cyt c. The phonon coupling was greater for Zn cyt c than for porphyrin cyt c, which may be due to the extra coupling to the polypeptide chain by metal ligation.

The mechanism of action of allosteric effectors in various types of hemoglobins is still not completely understood. It is known from time-dependent structural studies, and computer simulations, that not only certain structural changes are important in this phenomenon, but also the complex changes in the dynamics of the molecular assembly.In this study we report data on the high-pressure compressibility of human hemoglobin. We used pressure tuning combined with fluorescence line narrowing to estimate the compressibility of the alpha and beta subunits in human hemoglobin A. In each experiment in either the alpha or the beta subunits the heme was Fe: Zn-substituted to enable fluorescence spectroscopy.We have found that the compressibility of the two subunits are different, the beta having a larger, approximately 0.3 1/GPa compressibility, while the alpha subunits have approximately 0.1 1/GPa. Surprisingly, the allosteric effectors cause a significant reduction of the compressibility in the beta subunits, while no significant change was observed in the alpha subunits.This asymmetry in the compressibility values points to an asymmetry in the dynamics of the two subunits. Moreover, the asymmetry in the response to allosteric effectors suggests a distinct role of the beta subunits in the response sequence leading to changes in the oxygen binding capability.