Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

Low levels of irradiation modify lipid domains in model membranes: a laser Raman study

We have used Raman spectroscopy to study the effects of ionizing radiation on thermal transitions of dipalmitoyl lecithin + polyunsaturated fatty acid liposomes. Raman spectra in the CH (2800-3000 cm-1), C = C (1600-1680 cm-1), and C-C (1000-1150 cm-1) stretching regions are sensitive to ionizing radiation. The CH stretching of acyl chains yields three strong bands around 2850, 2880, and 2930 cm-1. The ratios of the relative intensities of 2880 and 2850 cm-1 bands, i.e., I2880/2850, when plotted against temperature show multiple infection points which correspond to multiple spectroscopic transitions. These are ascribed to a separate phase with distinctive proportions of lecithin and polyunsaturated fatty acids. We find these transitions sensitive to low levels of ionizing radiation. Doses as low as 5-15 rad after 48 h of 60Co gamma irradiation and 60 kVp X irradiation drastically broaden and shift the polyunsaturated rich phase which occurs at lower temperatures (-7 to +5 degrees C) than that of pure dipalmitoyl lecithin (39 degrees C). In addition a new transition around 46 degrees C also emerges upon irradiation (48 h postirradiation). These irradiation effects can be accelerated by the presence of catalytic amounts of Fe2+/EDTA +H2O2. The membrane transition modification is more sensitive to 60 kVp X rays in comparison to 60Co gamma rays owing to the high LET component of the former. The intensity of 1660 cm-1 band, assigned to C = C stretching in the cis-configuration, loses intensity upon irradiation. Concomitantly, a new band around 1675 cm-1, assigned to trans-configuration, emerges. Similarly the increase in the "order parameter" as calculated from the relative intensities of C--C stretching bands indicates rigidification of membrane. Various factors such as reduction in unsaturation, increase in trans-configuration, and the formation of multiple peroxidation products are invoked as lipid phase modifiers.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

Aggregation of chymotrypsinogen: portrait by infrared spectroscopy.

Changes in the secondary structure and aggregation of chymotrypsinogen were investigated by infrared difference spectroscopy in conjunction with temperature and pressure tuning IR spectroscopy; both the amide I' band and side chain bands were studied. A prominent component of the amide I' band in the difference spectrum obtained upon cooling a chymotrypsinogen solution, or increasing the hydrostatic pressure, was observed in the region between 1627 and 1622 cm-1. Under denaturing conditions a white gel was formed, which is attributed to irreversible self-association or aggregation. This process was accompanied by the appearance of two new amide I' bands in the infrared spectrum of the protein: a very strong band at 1618 cm-1 and a weak band at 1685 cm-1. These bands are assigned to peptide segments with anti-parallel aligned beta-strands.     

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.     

Studies on bovine serum amylase; evidence for two loci

Two genetically controlled polymorphic systems of amylase enzymes are described in sera from Friesian, Jersey, Guernsey, Hereford, Ayrshire and Charolais cattle. One of these Ami has been described previously and is controlled by two codominant alleles in British cattle, AmI ^B and AmI ^C. Evidence presented here suggests that it could be a gamma.amylase. The other enzyme which is described here for the first time has many of the properties of an alpha amylase. It is also polymorphic and controlled by two codominant alleles AmII ^A and AmII ^B. AmII ^A is only present in low frequency. The inheritance of both enzyme systems has been studied in 419 families.     

Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy.

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.     

An infrared spectroscopic study of the interactions of carbohydrates with dried proteins

Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

Fourier transform infrared study of proteins with parallel beta-chains

Deconvolved and second derivative Fourier transform infrared spectra of the proteins flavodoxin and triosephosphate isomerase have been obtained in the 1600 to 1700 cm-1 (amide I) region. To our knowledge these results provide the first experimental infrared data on proteins with parallel beta-chains. Characteristic absorption bands for the parallel beta-segments are observed at 1626-1639 cm-1 (strong) and close to 1675 cm-1 (weak). Previous theoretical studies based on hypothetical models with large, regular beta-sheets had suggested bands close to 1650 and 1666 cm-1. Our new assignments were confirmed by band area measurements, which yield conformational information in good agreement with results from X-ray diffraction data. The spectra were compared with corresponding spectra of concanavalin A and carboxypeptidase A. The first contains only antiparallel beta-segments, the second "mixed" beta-segments, with some strands lying antiparallel and others parallel. None of the observed amide I band frequencies assigned to parallel beta-chains occurs in the 1650 cm-1 region associated with helical segments.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Quantitative determination of hydrocarbon chain conformational order in bilayers of saturated phosphatidylcholines of various chain lengths by Fourier transform infrared spectroscopy

The infrared spectra of aqueous dispersions of a homologous series of symmetric-chain, disaturated phosphatidylcholines, with fatty acyl chain lengths ranging from 12 to 19 carbons, have been measured at comparable reduced temperatures in their liquid-crystalline phases. The infrared spectra of these compounds contain bands that are dependent on the conformation of the fatty acyl chains. In particular, in the 1400-1300-cm-1 spectral region, there are bands due to CH2 wagging which are specific for the different types of gauche conformers. Thus, gauche-trans-gauché sequences (or kinks) give a band at 1367 cm-1, end-gauche conformers a band at 1341 cm-1, and double-gauche conformers a band at 1355 cm-1. The intensities of these bands were determined and normalized to the intensity of the conformation-insensitive band due to symmetric methyl bending at 1378 cm-1. The intensities of the different "gauche" bands yield a "per chain" intensity, which is directly related to the concentration of the different types of conformational defects. We find that, within experimental error, the concentration of end-gauche and double-gauche conformers is relatively low and practically invariant with chain length when a series of homologous phosphatidylcholines are compared at the same reduced temperature. In contrast, the concentration of gauche-trans-gauché sequences (kink defects) is much higher and increases as the chain length increases. For dipalmitoylphosphatidylcholine we find that there are about 1.2 kink, 0.5-0.6 end-gauche, and 0.4 double-gauche conformers per hydrocarbon chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin

A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.     

Components of the carbonyl stretching band in the infrared spectra of hydrated 1,2-diacylglycerolipid bilayers: a reevaluation.

Previous vibrational spectroscopic studies of solid acyl-alkyl and diacyl phosphatidylcholines suggested that the sn1- and sn2-carbonyl stretching modes of 1,2-diacylglycerolipids have different absorption maxima. To address the assignment of sn1- and sn2-carbonyl stretching modes of hydrated 1,2-diacylglycerolipids, aqueous dispersions of 1-palmitoyl-2-hexadecyl phosphatidylcholine (PHPC), 1-hexadecyl-2-palmitoyl phosphatidylcholine (HPPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), as well as hydrated samples of unlabeled, sn1-13C=O-labeled, sn2-13C=O-labeled, and doubly 13C=O-labeled dimyristoylphosphatidylcholine (DMPC) were examined by Fourier transform infrared spectroscopy. The ester carbonyl stretching (nu C=O) bands of HPPC and PHPC each exhibit maxima near 1726 cm-1 and appear to be a summation of three subcomponents with maxima near 1740 cm-1, 1725 and 1705-1711 cm-1. In contrast, the nu C=O band of DPPC exhibits its maximum near 1733 cm-1 and appears to be a summation of two components centered near 1742 and 1727 cm-1. Thus the ester carbonyl group of the acyl-alkyl PCs appears to reside in a more polar environment than the ester carbonyl groups of their diacyl analogue. This observation implies that the polar/apolar interfaces of hydrated bilayers formed by PHPC and by HPPC are significantly different from that of DPPC and raises the question of whether the acyl-alkyl PCs are suitable models of their diacyl analogue. The absorption maximum of the nu C=O band of the doubly 13C=O-labeled DMPC occurs near 1691 cm-1 and those of its subcomponents occur near 1699 and 1685 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of specifically 13C=O labeled phosphatidylcholine multilayers from polarized attenuated total reflection FT-IR spectroscopy.

Oriented multilayers of 1-myristoyl-2(1-13C)-myristoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DMPC) and 1-palmitoyl-2(1-13C)-palmitoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DPPC) were investigated by use of attenuated total reflection infrared spectroscopy with polarized light. Experiments were performed with the aim to determine the orientation of the two ester groups in these phospholipids in the solid state and in the hydrated state at temperatures below and above the respective gel to liquid-crystalline phase transitions. Substitution of the naturally occurring 12C carbonyl carbon atom by 13C in the ester group of the sn-2 chain of DMPC and DPPC shifts the infrared absorption of the carbonyl double bond stretching vibration to lower frequency. This results in two well-resolved ester C=O bands which can be assigned unequivocally to the sn-1 and sn-2 chains as they are separated by more than 40 cm-1. The two ester CO-O single bond stretching vibrations of the molecular fragments-CH2CO-OC-are also affected and the corresponding infrared absorption band shifts by 20 cm-1 on 13C-labeling of the carbonyl carbon atom. From the dichroic ratios of the individual ester bands in 2(1-13C)DMPC and 2(1-13C)DPPC we were able to demonstrate that the sn-1 and sn-2 ester C=O groups are similarly oriented with respect to the bilayer plane, with an angle greater than or equal to 60 degrees relative to the bilayer normal. The two CO-O single bonds on the other hand have very different orientations. The CH2CO-OC fragment of the sn-1 chain is oriented along the direction of the all-trans methylene chain, whereas the same molecular segment of the sn-2 carbon chain is directed toward the bilayer plane. This orientation of the ester groups is retained in the liquid-crystalline phase. The tilt angle of the hydrocarbon all-trans chains, relative to the membrane normal, is 25 degrees in the solid state of DMPC and DPPC multibilayers. In the hydrated gel state this angle varies between 26 degrees and 30 degrees, depending on temperature. Neither the orientation of the phosphate group, nor that of the choline group varies significantly in the different physical states of these phospholipids.     

Infrared spectroscopy of a single cell--the human erythrocyte

Methods for obtaining the infrared spectrum of a single erythrocyte by infrared microscopy have been developed. The spectrum contains the amide I, II, and III bands characteristic of protein secondary structure near 1650, 1550, and 1300 cm-1, respectively. Bound carbon monoxide exhibits a readily measured band at 1951 cm-1 for 12C16O and 1907 cm-1 for 13C16O. Both amide and CO bands are similar to those found for purified hemoglobin A. Spectra can be obtained in H2O or D2O media under physiologically relevant conditions. Single cell infrared spectroscopy (SCIR) permits the qualitative and quantitative determination of differences among individual red cells. These results suggest many potential applications for SCIR for the measurements of properties of individual cells at the molecular level under physiologically relevant conditions.     

Dynamic interactions of CO with a3Fe and CuB in cytochrome c oxidase in beef heart mitochondria studied by Fourier transform infrared spectroscopy at low temperatures

Carbon monoxide bound to cytochrome c oxidase has been observed by Fourier transform infrared spectroscopy between 10 K and 280 K in the dark and during and after continuous photolysis. CO bound to a3Fe absorbs near 1963 cm-1, with minor bands at lower frequencies. Photolysis at low temperatures transfers CO to CuB, with the major component near 2062 cm-1 and a minor one near 2043 cm-1. Vibrational absorptions are assigned by comparison with heme and copper carbonyls, by frequency dependence of all bands on the isotopic mass of CO, and by similar behavior of major and minor components with photolysis and relaxation kinetics as a function of temperature. Reformation of a3FeCO after photolysis is an apparent first order process below 210 K with a distribution of rate constants. The kinetics are well described by a power law. Arrhenius behavior is followed between 140 K and 180 K to yield a peak activation enthalpy of 40.3 kJ/mol and a distribution in g(H) = 2.56 kJ/mol (full width at half-maximum). The major component of a3FeCO shows a very narrow CO absorption band (full width at half-maximum = 2.4 cm-1), while that of CuBCO shows a broader CO absorption (full width at half-maximum = 6 cm-1). These data indicate that in the reduced carbon monoxide complex, a3FeCO is in highly ordered nonpolar surroundings sufficiently separated from CuB that it is not perturbed by motion of the latter, while CuBCO is in less ordered, more flexible surroundings.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

Zymographic characterization of bacteriolytic enzymes produced by oral streptococci

Zymographic analysis was performed to know the bacteriolytic enzyme profiles of 4% SDS extracts of oral streptococci, Streptococcus mutans, S. sobrinus, S. sanguis, S. mitis and S. salivarius. We investigated the five strains in each species and found that the profile was very similar among strains of the same species except for S. salivarius(the profile was classified into two types). On the other hand, the profile was considerably different among species. Two major bacteriolytic enzymes of S. mutans showing molecular mass of 80 and 100 kDa were found using SDS-boiled S. mutans or S. sobrinus cells as substrate. These bacteriolytic activities were less apparent in the gel containing S. mitis or S. salivarius, and also not detectable in the gel containing S. sanguis. S. sobrinus extract showed only one bacteriolytic band (78 kDa) as strong activity using S. sobrinus cells as substrate. S. sanguis extract showed no bacteriolytic bands using any streptococcal cells. Extracts of either S. mitis or S. salivarius showed weak activity by using respective strains as substrate.     

Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structures

Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.