Radial packing, order, and disorder in collagen fibrils.

Collagen fibrils resemble smectic, liquid crystals in being highly ordered axially but relatively disordered laterally. In some connective tissues, x-ray diffraction reveals three-dimensional crystallinity in the molecular packing within fibrils, although the continued presence of diffuse scatter indicates significant underlying disorder. In addition, several observations from electron microscopy suggest that the molecular packing is organized concentrically about the fibril core. In the present work, theoretical equatorial x-ray diffraction patterns for a number of models for collagen molecular packing are calculated and compared with the experimental data from tendon fibrils. None of the models suggested previously can account for both the crystalline Bragg peaks and the underlying diffuse scatter. In addition, models in which any of the nearest-neighbor, intermolecular vectors are perpendicular to the radial direction are inconsistent with the observed radial orientation of the principal approximately 4 nm Bragg spacing. Both multiple-start spiral and concentric ring models are devised in which one of the nearest-neighbor vectors is along the radial direction. These models are consistent with the radial orientation of the approximately 4 nm spacing, and energy minimization results in radially oriented crystalline domains separated by disordered grain boundaries. Theoretical x-ray diffraction patterns show a combination of sharp Bragg peaks and underlying diffuse scatter. Close agreement with the observed equatorial diffraction pattern is obtained. The concentric ring model is consistent with the observation that the diameters of collagen fibrils are restricted to discrete values.     

Synthesis and physical properties of (hydroxyproline-proline-glycine)10: Hydroxyproline in the X-position decreases the melting temperature of the collagen triple helix

The collagen-like polytripeptide (hydroxyproline-proline-glycine)₁₀ was synthesized with a solid-phase procedure. Analytical ultracentrifugation indicated that the peptide in aqueous solution at 6 °C had a molecular weight of 2550, the expected size of a single chain. The peptide had a relatively small negative optical rotation at 578 nm, and it did not show a thermal transition as is seen with collagen or collagen-like polytripeptides which form triple helices. At low temperatures in aqueous solution, the circular dichroism spectrum was similar to that of triple-helical collagen and collagen-like peptides in that there was a positive peak at 224 nm and a negative peak at 200 nm. The amplitudes of the peaks, however, were considerably less than the peaks obtained with triple-helix proteins and peptides. Since (proline-proline-glycine)₁₀ was triple helical under the same conditions, the results demonstrated that hydroxyproline in the X-position of the repeating -glycine-X-Y- sequences decreases rather than increases, the thermal stability of the triple helix. This positional specificity cannot be explained by any of the current models for the structure of the triple helix or any of the current proposals for how hydroxyproline stabilizes the structure.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

The thermal behavior of collagen in solution: effect of glycerol and 2-propanol

The thermal stability of different solutions of collagen (Col), collagen mixed with glycerol (Col-G) and collagen mixed with 2-propanol (Col-P) was studied by differential scanning calorimetry (DSC), viscosity and fluorescence. The DSC and viscosity methods showed that glycerol increased the denaturation temperature of collagen about 2°C, while 2-propanol decreased it about 2°C. The values of intrinsic viscosity ([η]) for Col, Col-G and Col-P were 21.67, 20.20 and 24.71 dl/g, respectively. Huggins coefficient (k(H)) increased in the presence of glycerol and decreased in the presence of 2-propanol. It was suggested that glycerol promoted the dissolution of collagen molecular aggregates while 2-propanol enhanced the aggregation. Fluorescence spectra were investigated within the temperature ranging from 15 to 45°C. By comparing the sign of peaks in the two-dimensional (2D) fluorescence correlation maps, the orders of peak response were ～360, ～410>297 nm for Col and Col-G, and 297>～360, ～410 nm for Col-P, respectively. These indicated that the respondences of tyrosine residues, excimer-like species and bityrosine on the perturbation of temperature were different in the presence of glycerol and 2-propanol.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

Neutron diffraction studies of collagen in fully mineralized bone

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.     

Synchrotron ultraviolet microspectroscopy on rat cortical bone: involvement of tyrosine and tryptophan in the osteocyte and its environment

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately.     

Circular dichroism of rhodopsins and porphyropsins

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

Fluorescent-labeled collagen: Age-related differences and fluorescence changes during the lag phase of fibril formation

Acid-soluble collagens isolated from young and old rat tail tendon were fluorescent-labeled with dansyl hydrazine, which is capable of reacting with aldehyde groups in collagen. The dansyl fluorescence of aged collagen exhibited a weak peak at 525 nm, whereas that of young collagen had a stronger broad peak at 500 nm. Fibril formation in vitro was partially inhibited in these dansylated collagens. During the turbidity lag phase, the dansyl fluorescence was found to increase (30–50%), also shifting to 485 nm. These changes reveal the telopeptide conformation changes occurring during this period. A new fluorescence peak at 420 nm also increased during fibril formation. When the dansylated collagen was irradiated in air with uv light (340 nm), a rapid decrease of the dansyl fluorescence with a concurrent shift to 490 nm occurred. Also, the formation of fibrils was further inhibited. With increasing temperature, the dansyl fluorescence of young collagen decreased, whereas that of old collagen substantially increased, particularly at the denaturation temperature around 38°C. After denaturation, both fluorescences became similar in their intensity and position (490 nm). These findings are discussed in connection with both age-related structural changes of collagen and the mechanism of fibril formation.     

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.     

The estimated elastic constants for a single bone osteonal lamella

Micromechanical estimates of the elastic constants for a single bone osteonal lamella and its substructures are reported. These estimates of elastic constants are accomplished at three distinct and organized hierarchical levels, that of a mineralized collagen fibril, a collagen fiber, and a single lamella. The smallest collagen structure is the collagen fibril whose diameter is the order of 20 nm. The next structural level is the collagen fiber with a diameter of the order of 80 nm. A lamella is a laminate structure, composed of multiple collagen fibers with embedded minerals and consists of several laminates. The thickness of one laminate in the lamella is approximately 130 nm. All collagen fibers in a laminate in the lamella are oriented in one direction. However, the laminates rotate relative to the adjacent laminates. In this work, all collagen fibers in a lamella are assumed to be aligned in the longitudinal direction. This kind of bone with all collagen fibers aligned in one direction is called a parallel fibered bone. The effective elastic constants for a parallel fibered bone are estimated by assuming periodic substructures. These results provide a database for estimating the anisotropic poroelastic constants of an osteon and also provide a database for building mathematical or computational models in bone micromechanics, such as bone damage mechanics and bone poroelasticity.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

In vitro Synthesis of Native,Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.     

Biochemical and physiochemical characterization of pepsin-solubilized type-II collagen from bovine articular cartilage.

Solubilization of collagen from bovine articular with pepsin requires the preliminary extraction of proteoglycans from the ground substance. Biochemical and physiochemical properties of this pepsin-solubilized collagen are independent of the pretreatment (extraction with 1.5M-CaCl2, 5M-guanidinium chloride or 0.2M-NaOH) and of the age range (2-4-year-old and 2-month-old animals). Characterization of the de-natured components, of the CNBr peptides and of the amino acid and cross-link composition shows that the collagen of the hyaline cartilage is all type II. Electrical birefringence measurements showed the presence of tropocollagen molecules (length 280nm) and molecules whose length is slightly less than twice that of the tropocollagen molecules. This latter molecule may be a dimer composed of two monomers linked by intermolecular head-to-tail bonds and whose theoretical length (530nm), according to the quarter-stagger theory, is in good agreement with our measured values (510-530nm). We have verified that the beta-components of this collagen are formed of two alpha-chains linked by the stable intermolecular bond, dehydrodihydroxylysinonorleucine. These dimeric molecules are absent from solutions of skin collagen whose beta-components possess only aldol-type intramolecular cross-links. Although reconstituted fibres from solutions of skin and cartilage collagen are similar, the segment-long spacing crystallites formed with pepsin-solubilized cartilage collagen present a symmetrical and dimeric form corresponding to the lateral aggregation of two monomers with an overlap (90nm) of the C-terminal ends.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Characterization of Acid- and Pepsin-Soluble Collagens from the Cuticle of <Emphasis Type="Italic">Perinereis nuntia</Emphasis> (Savigny)

To extend the practical applications of collagen, alternatives to mammalian sources are needed. In this study, acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the cuticle of Perinereis nuntia (Savigny), and their physicochemical features and structures were examined. The yields of ASC and PSC were 3.89% and 6.74%, respectively. The glycine contents of both collagens accounted for approximately one-third of the total amino acid residues, and the sum totals of proline and hydroxyproline in ASC and PSC were 212 and 214 residues/1000 residues, respectively. However, the proline hydroxylation rates of ASC and PSC were 84.0% and 83.6%, respectively. The maximum absorption peaks of both ASC and PSC were detected at 233 nm. Zeta potential studies indicated that ASC and PSC have a net zero charge at pH 4.89 and 4.95, respectively. Fourier-transform infrared spectroscopy, circular dichroism, and X-ray diffraction confirmed the triple helical structure of the collagen. The denaturation temperatures (T_d) of ASC and PSC were 36.5 °C and 33 °C, respectively. Moreover, the collagens appeared to be loose, fibrous, and porous by scanning electron microscopy. These results suggested that collagen from the cuticle of Perinereis nuntia (Savigny) has potential commercial applications in the food, nutraceutical, and pharmaceutical industries.     

激光照射血液荧光光谱的初步研究

采用ＯＭＡ—ＩＩ微弱信号检测系统研究了人血液荧光光谱在激光照射下的变化情况。结果表明：在６３２．８ｎｍＨｅＮｅ激光诱导下,不同血液在６７０ｎｍ,７３０ｎｍ,９８１ｎｍ附近出现三个荧光峰;荧光强度在一定范围内与照射激光功率呈线性变化关系;随着激光照射时间的增加,三个峰位上的荧光强度下降,８分钟后趋于稳定值;在激光照射过程中,三个峰位出现不同数值的移动,同时在６７０ｎｍ和７３０ｎｍ两个荧光峰之间出现了竞争。     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Stretching type II collagen with optical tweezers

Collagens are the most abundant proteins of vertebrates and they provide mechanical and supportive functions in a wide range of connective tissues. Knowledge of the mechanical properties of single collagen molecules is essential in studying the self-assembly of collagen, the interaction between cells and extracellular matrix, the etiology of tissue degeneration and mechanism of regeneration, and the relationship between the structures and mechanical properties of tissues. Here we stretched single type II collagen molecules in neutral pH solution using optical tweezers. The molecular parameters of collagen were obtained by fitting force-extension curves into worm-like chain elasticity model. The molecule length of type II collagen monomer was 295.8 nm. The persistence length of type II collagen monomer was 11.2 nm. These observations indicate that collagen molecules are flexible rather than rigid rod molecules at neutral pH solution.