Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

The intermolecular space of reconstituted collagen fibrils

The extent, geometry and heterogeneity of the intermolecular space of hydrated, purified and reconstituted steer skin collagen fibrils has been characterized. The extent of the space has been assessed experimentally by an X-ray diffraction method and a new physical chemical technique, and found to be 1.14 ml per gram collagen. A theoretical model relating the intermolecular space to X-ray diffraction parameters has been presented, and this suggests that the geometry of the intermolecular space arises from a near-hexagonal packing of the collagen molecules. On the basis of an assumed microfibrillar packing model and a geometric construction of the shape of a collagen molecule, the distribution of the space within reconstituted collagen fibrils has been characterized as follows: 0.13 ml of the intermolecular space/g collagen can be attributed to the helical groove of the collagen molecules per se and 1.01 ml/g is interstitial; 0.66 ml/g is present in the form of “pores” (hexagonally-closed packed spaces), whereas 0.48 ml/g is present in the form of “holes” (hexagonal volume defects); 0.73 ml/g of the intermolecular space is associated with a region of the collagen fibrils where holes are localized and 0.41 ml/g is attributable to the regions of the fibril in which pores only are present.     

Chromosomal banding patterns produced by methyl green-pyronin staining after trypsin treatment.

A method is described for producing banding patterns with methyl green-pyronin (MGP) stain in chromosomes of fibrosarcoma cells. 1) The stain was made by mixing equal volumes of 2% aqueous pyronin G, 2% aqueous methyl green, distilled water, and 0.1 M acetate buffer (pH 5.7). 2) Treatment with colcemide and hypotonic KCl (0.075 M) was performed as usual. 3) Metaphase chromosomes were prepared using the flame-drying technique and treated with 0.25% trypsin at 37 C for 45 to 90 seconds. Before staining, the slides were rinsed in PBS, in distilled water, and then were dipped in 0.05 M acetate buffer. 4) Chromosomes were stained for more than 20 minutes, rinsed in distilled water, and hot-air dried. Satisfactory results were obtained in uncontracted metaphase chromosomes. MGP stain has the advantage of permitting much longer trypsin treatment and staining time than the trypsin-Giemsa method while providing satisfactory banding patterns.     

A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils

This report describes an assay for measuring the activity of collagenases using reconstituted fibrils of fluorescein-labeled soluble collagen as substrate. The labeling of commercially available material is easy and not expensive. The assay is very sensitive, reproducible, and is linear with collagenase concentration up to 100% consumption of the reconstituted fibrils. Readings of collagenolytic activity can be determined directly by measuring the fluorescence of the released labeled peptides without further processing of the data.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Reconstituted collagen fibrils. Fibrillar and molecular stability of the collagen upon maturation in vitro.

During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal stability of collagen increased and the pepsin-solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in a biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen.     

The staining pattern of collagen fibrils. Improved correlation with sequence data.

The periodic banding pattern of stained collagen fibrils observed in the electron microscopic can be correlated with the charge distribution deduced from the amino acid sequence. Earlier work used alpha 1 chain sequence data only. The present study incorporates alpha 2 as well as alpha 1 sequence data, so that the complete distribution of charged residues is used. Correlation is improved if it is supposed that the extrahelical terminal regions are contracted. The optimal value of the periodicity, D, (previously 232.3 +/- 0.5 residues using alpha 1 data only), is now 234.2 +/- 0.5 residues, assuming uniform spacing of residues in the helical body of the molecule. This value agrees better with values obtained by others from analyses of interactions between molecules, using sequence data alone. Using the improved value of D, the relative axial locations of the charged residues in the fibril are displayed. In this way, the charged residues contributing to each band in the fibril staining pattern can be identified.     

An electrostatic model for collagen fibrils. The interaction of reconstituted collagen with Ca++, Na+, and Cl−

A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å. The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect. The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M^?1, respectively. The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties. The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.     

Thermodynamic studies of the assembly in vitro of native collagen fibrils

Measurements of the solubility of calf-skin tropocollagen in neutral phosphate buffers in the temperature range 20-37 degrees C show that native collagen fibril formation is an endothermic process made thermodynamically favourable by a large positive entropy of precipitation associated with structural changes in the surrounding solvent. The effect of inorganic ions and small solute molecules on precipitation seems to be correlated with their structural effects on liquid water. Heterogeneity in the precipitation properties of the collagen solutions may be related to changes in the configurational entropy of the macromolecules due to intramolecular cross-linking.     

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.