Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions.

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent.     

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.     

Changes in Raman vibrational bands of calf thymus DNA during the B-to-A transition

The B -to-A conformational transition of calf thymus DNA fibers was followed employing Raman spectroscopy. The transition was induced by soaking DNA fibers in water/ethanol mixtures increasing from 60 to 85% ethanol (v/v). Intensity changes of 17 Raman vibrational bands were quantified in the region from 400 to 860 cm^?1. Two bands at 500 and 784 cm^?1 were employed as internal standards. These bands do not appear to change in intensity with ethanol concentration. Large intensity changes relative to these two bands are observed between 70 and 74% ethanol for backbone vibrations at 708, 808, and 835 cm^?1, and base vibrations at 682, 730, and 750 cm^?1. These results indicate that a highly cooperative conformational change takes place between different portions of DNA in the B -to-A transition. Relative intensity changes preceding the onset of the major transition are observed in only two bands; at 835 cm^?1, assigned to a ribose–phosphate vibration, and at 750 cm^?1, assigned to thymine. The implications of these pretransition changes are discussed.     

Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations.

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.     

Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-²H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H₂O and 1.05 ± 0.03 for ²H₂O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.     

二氧化硅与脂质体相互作用的激光拉曼光谱研究

脂质体在SiO_2作用下1127cm~(-1)与1093cm~(-1)强度比和2883cm~(-1)与2847cm~(-1)强度比均降低,715cm~(-1)谱带强度也降低.并且频率有2—3cm~(-1)的位移,峰形变宽.表明磷脂以其极性头部与SiO_2结合,形成稳定的复合体.磷脂头部的作用和脂质体的变形引起烃链构象变化.TiO_2不引起拉曼光谱的变化,与脂质体的作用甚微.     

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering.

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.     

Laser Raman study of internally perfused muscle fibers. Effect of Mg2+, ATP and Ca2+

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca2+, Mg2+ and ATP. The comparison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg2+ and Ca2+ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg2+ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca2+ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the alpha-helical content, or of the orientation, of some of the contractile proteins.     

Conformational dependence of the Raman scattering intensities from polynucleotides

The intensity of Raman scattering from the various Raman active vibrations of poly-(riboadenylic acid), poly(ribocytidylic acid), poly(ribouridylic acid), and poly(riboinosinic acid) in moderately dilute solutions were examined as the temperature was changed to alter their conformation. It was found that certain highly intense, highly polarized Raman bands from the totally symmetric, i.e., in-plane, ring vibrations of the nucleic acid bases become less intense as the chains become more ordered in solution. Since these vibrations occur at frequencies which are markedly different for each type of base, Raman spectroscopy appears to provide a new method for the characterizing of the average conformation of each of the bases in solution. A theory for the resonant Raman effect is given in which it is shown that, a decrease in resonant Raman intensity is to be expected if one obtains a decrease in the intensity of the corresponding ultraviolet absorption band with which the incident light is resonant. If it is assumed that certain Raman bands derive their intensity predominantly from the first few ultraviolet absorption intensities, then a qualitative explanation of our observed conformational dependence of the ordinary Raman intensities can be obtained.     

Infrared and Raman spectra of phosphatidyl-ethanolamine and related compounds.

Infrared and Raman spectra of phosphatidylethanolamine from Escherichia coli, L-alpha-glycero-phosphorylethanolamine and 0-phosphorylethanolamine were obtained. Most of the bands were assigned to each vibrational mode based on the N deuteration effect, comparison of the intensity in the infrared and Raman spectra and the depolarization degree measurement in the Raman spectra. The spectra of phosphatidylethanolamine can be interpreted by assuming that the molecule takes the dipolar ionic structure both in non-polar solvent and in solid.     

Deciphering the finger Prints of Brain Cancer Astrocytoma in comparison to Astrocytes by using near infrared Raman Spectroscopy

To explore the biochemical differences between brain cancer cells Astrocytoma and normal cells Astrocyte, we investigated the Raman spectra of single cell from these two cell types and analyzed the difference in spectra and intensity. Raman spectrum shows the banding pattern of different compounds as detected by the laser. Raman intensity measures the intensity of these individual bands. The Raman spectra of brain cancer cells was similar to those of normal cells, but the Raman intensity of cancer cells was much higher than that of normal cells. The Raman spectra of brain cancer Astrocytoma shows that the structural changes of cancer cells happen so that many biological functions of these cells are lost. The results indicate that Raman spectra can offer the experimental basis for the cancer diagnosis and treatment.     

The study of microviscosity of plasma membranes of Nitella cells during rest and excitation

Changes in the microviscosity of excitable membranes was investigated using resonance Raman spectroscopy of carotenoids. The Raman resonance spectra of carotenoids in Nitella cells were excited by 514.5 nm line of an argon ion laser. The bands at 1525 cm-1, 1160 cm-1 and 1008 cm-1 were observed and they were assigned to C=C, C-C and C-CH vibrations, respectively. The rhythmic excitation of cell reduced the intensity and increased the ratios of intensity of major carotenoid bands with no noticeable shift in the position of peaks. The Arrhenius plot of relative intensity ratios of 1525 cm-1 and 1160 cm-1 bands versus reciprocal temperature showed a change of the slope in the range of 13-18 degrees C. This indicates a membrane phase transitions in which a reorientation of carotenoids species takes place. The interpretation was supported by parallel microcalorimetric and EPR measurements. The decrease of microviscosity with increasing temperature is probably caused by changes in polyene chain conformation. It is suggested that membrane microviscosity during NH4(+)-stimulated rhythmic excitation of algal cells increases, and membrane-associated carotenoids act as microviscosity-sensitive "potential sensor" for the channel.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

Effects of endurance exercise-training on single-fiber contractile properties of insulin-treated streptozotocin-induced diabetic rats.

The purpose of this study was to characterize the contractile properties of individual skinned muscle fibers from insulin-treated streptozotocin-induced diabetic rats after an endurance exercise training program. We hypothesized that single-fiber contractile function would decrease in the diabetic sedentary rats and that endurance exercise would preserve the function. In the study, 28 rats were assigned to either a nondiabetic sedentary, a nondiabetic exercise, a diabetic sedentary, or a diabetic exercise group. Rats in the diabetic groups received subcutaneous intermediate-lasting insulin daily. The exercise-trained rats ran on a treadmill at a moderate intensity for 60 min, five times per week. After 12 wk, the extensor digitorum longus and soleus muscles were dissected. Single-fiber diameter, Ca(2+)-activated peak force, specific tension, activation threshold, and pCa(50) as well as the myosin heavy chain isoform expression (MHC) were determined. We found that in MHC type II fibers from extensor digitorum longus muscle, diameters were significantly smaller from diabetic sedentary rats compared with nondiabetic sedentary rats (P < 0.001). Among the nondiabetic rats, fiber diameters were smaller with exercise (P = 0.038). The absolute force-generating capacity of single fibers was lower in muscles from diabetic rats. There was greater specific tension (force normalized to cross-sectional area) by fibers from the rats that followed an endurance exercise program compared with sedentary. From the results, we conclude that alterations in the properties of contractile proteins are not implicated in the decrease in strength associated with diabetes and that endurance-exercise training does not prevent or increase muscle weakness in diabetic rats.     

Raman pH profiles for nucleic acid constituents I. Cytidine and uridine ribonucleosides.

Raman spectra of aqueous solutions of uridine and cytidine have been recorded as a function of pH with the band intensities and vibrational frequencies monitored to determine bands which may be considered as diagnostic of the concentration of the various species. Quantitative band intensity measurements indicate that not all pH-sensitive bands can be considered as diagnostic of the pK value for the acid form of the nucleoside, and for the percent species in solution. Although the accuracy of the Raman band intensity method is inherently less than that of the titrimetric or visible-ultraviolet spectrophotometric methods, the pK values and percent species agree well with those obtained from these methods. The utility of the results obtained from the pH profiles for cytidine is discussed in terms of the effect of acidification on the structural and conformational characteristics of polycytidylic acid in solution.     

Fatty acids,part X. Raman spectra of all dimethylene interrupted methyl cis,cis-octadecadienoates and octadecadiynoates

The Raman spectra of all the dimethylene interrupted methyl cis, cis-octadecadienoates and octadecadiynoates have been studied. The Raman band positions and their relative intensities for the ν(CC), ν(CC), ν(CO), ν(CH) and δ(CH₂) modes are recorded. The height intensity of the bands arising from ν(CC) relative to ν(CO) provides a means of determining the number of cis-ethylenic bonds in a mono-ester. In the acetylenic series, the intensity of the bands arising from ν(CC) relative to ν(CO) failed to indicate with certainty the number of acetylenic bonds in the mono-esters studied, due to the weak intensity of the band due to ν(CO). However a better correlation between the relative intensities of the ν(CC) and δ(CH₂) bands is established instead. An attempt to correlate the areas under the bands due to ν(CC), (CC), (CO) and δ(CH₂) failed to produce any significant results. The Raman spectra of the methyl octadec-cis-10-en-5-ynoate and methyl octadeca-5, 10-diynoate are also recorded.     

Calcium and pH-induced structural changes in skinned muscle fibers: prevention by N-ethylmaleimide.

Optical diffractometry was used to investigate structural changes in contractile proteins from chemically skinned muscle fibers. The intensity of the first order diffraction line decreased in response to calcium and alkaline pH. Fibers pre-treated with sulfhydryl blocking agents did not exhibit the calcium- or pH- induced intensity decreases. We suggest that the decreases in intensity result from structural changes in the thick filament. Optical diffractometry may be a useful technique for the investigation of conformational changes in myosin.     

ELECTRON MICROSCOPIC AND HISTOCHEMICAL OBSERVATIONS OF MUSCLE DEGENERATION AFTER TOURNIQUET

As an experimental model for the different forms of muscle degeneration, injury caused by 2 hours' ischemia has been studied from 20 minutes to 16 hours after release of the tourniquet. Discoid degeneration developed in stretched fibers by dissolution of the I bands (Z substances and actin). The discs represented the Q bands (A-H-A). In fibers which passively maintained contraction lengths during degeneration, the Z substances were dissolved, but the continuity of the fibrils was preserved, since the filaments are continuous over all sarcomeres under these conditions. Mitochondria and the tubules of the endoplasmic reticulum swelled, ruptured, and disintegrated. Granular degeneration developed in fibers where mitochondria were abundant. Unstretched degenerating fibers with few mitochondria gave a homogeneous or hyaline appearance. The different forms of degeneration therefore were dependent on the status of stretch and the fiber type. The extent of degeneration was not a function of time after ischemia, there being both nearly normal and severely damaged fibers at 20 minutes and 16 hours after the release of tourniquets. When degeneration occurred, however, the basic alterations were the same in all fibers; there was mitochondrial and reticular swelling, dissolution of the Z substances, and finally disintegration of the contractile material. Some damage developed in the sarcolemmas and capillaries. The mitochondrial disintegration was not linked with inactivation of the succinic dehydrogenase system.