Donald G. Wallace

Injectable collagen as a pH-sensitive hydrogel

Injectable collagen is a concentrated dispersion of phase-separated collagen fibres in aqueous solution. The structure and properties of collagen fibres are defined by the magnitudes of electrostatic and hydrophobic attractive forces between neighbouring collagen molecules within collagen fibres. The structure and mechanical properties of collagen fibre dispersions were studied by dynamic rheological measurements and by polarized microscopy. Rheological measurements were performed over pHs ranging from 6 to 9 and over temperatures ranging from 283 to 298 K. At higher pHs the fibre dispersions were found to possess more rigid fibres and stronger inter-fibre attractive forces. This response is argued to result from changes in the ionization of amino acid side chains, which result in larger net-electrostatic attractive forces. Raising the temperature caused fibres to rigidify through enhanced hydrophobic attractive forces. Gels formed by lower pH-higher temperature fibre dispersions possess different properties than gels formed at higher pHs and lower temperatures.

Albumin Differences Among Ranid Frogs: Taxonomic and Phylogenetic Implications

Wallace, D. G., M. C. King and A. C. Wilson (Biochemistry Dept., University of California, Berkeley, California 94720) 1973. Albumin differences among ranid frogs: taxonomic and phylogenetic implications. Syst. Zool., 22:1-13.-Since morphological analysis of living and fossil material has provided little information on taxonomic and phylogenetic relationships among species in the frog family Ranidae, an attempt was made to obtain such information by comparing their serum albumins. Rabbit antisera were prepared against the purified serum albumins of seven ranid species and tested by the miorocomplement fixation method for reactivity with the albumins of 36 ranid species. This method provides a measure of the degree of amino acid sequence difference between albumins. The taxonomic and phylogenetic schemes we have derived from the albumin comparisons are generally in agreement with nonmolecular evidence. In some cases, they go beyond it, permitting demarcations among taxa that were previously unresolved. For example, it is proposed that the North American species of Rana are divisible into at least two major subgroups; an eastern subgroup, which includes R. pipiemn and R. catesbeiana, and a western cluster, which includes R. aurora. The nearest relatives of the westem cluster appear from albumin comparisons to be the R. temporaria cluster of the Palearctic region. The albumin results also permitted the construction of a phylogenetic tree with a very low percent standard deviation. With the aid of this tree and the molecular clock hypothesis we have estimated the approximate divergence times of various groups, subgroups and clusters of Holarctic ranid species. The molecular differences among Rana species are at least as large as those usually found within orders of placental mammals. This raises an important question regarding the ranking of taxonomic categories. If the genus Rana is equivalent in terms of genetic dispersion to an order of mammals, should one elevate the rank of Rana to the ordinal level? We believe that the molecular findings provide taxonomists with a dilemma. [Rana; albumins; phylogeny.]

The effects of heparin on the physicochemical properties of reconstituted collagen.

Pepsin-solubilized bovine dermal collagen was reconstituted in 0.02 M sodium phosphate (pH 7.2), concentrated to 30-40 mg/ml, and adjusted to physiological ionic strength by addition of sodium chloride. These preparations, at 4-15 degrees C, are fibrillar suspensions composed of fibrils of varying diameters and nonassociated molecules. Addition of heparin to these suspensions promoted a dose-dependent increase in average fibril diameter as measured by turbidimetry and electron microscopic analyses. These effects were relatively specific for heparin and heparin-like glycosaminoglycans. Chondroitin sulfate and hyaluronic acid had little or no effect on fibrillar diameters under these conditions, whereas dermatan sulfate had an intermediate effect on fibrillar reorganization. Differential scanning calorimetry revealed that addition of optimal concentrations of heparin generated fibrils of higher stability and that this effect was associated with the disappearance of structures of lower stability, including nonassociated molecules and thin fibrils. Light microscopic analyses of the fibrillar collagen/heparin matrix showed it to be a more open network of distinct collagen fibers than was observed with the fibrillar collagen preparation alone. Binding experiments indicated that heparin bound to fibrillar collagen in a saturable fashion with a Kd of approximately 4 X 10(-7) M. Creep experiments provided evidence that the addition of heparin to fibrillar collagen suspensions greatly reduces the gelation phenomenon that is normally observed when such suspensions are warmed to 37 degrees C. These differences in fibrillar architecture may be in part responsible for differences noted in the biological response to fibrillar collagen and fibrillar collagen/heparin implants in vivo (McPherson et al., 1988).

Comparison of frog albumins with those of other vertebrates

SummarySerum albumin was purified from two frog species (Rana pipiens andR. catesbeiana) and compared by several physical, chemical and immunological techniques with the purified albumins of a reptile, a bird and three mammals. Although the frog albumins proved to be very similar to those of other vertebrates in physical and chemical properties, no immunological relationship was detected in direct precipitin and complement fixation tests. The absence of an immunological relationship between frog albumins and those of other vertebrates is expected because other evidence indicates that albumin evolves very rapidly. Fossil evidence indicates that the lineage leading to frogs separated from those leading to other vertebrates so long ago that we should expect frog albumins to have become more than 40% different in amino acid sequence from those of other vertebrates and thus to exhibit no cross reactivity in direct precipitin or complement fixation tests.

Biological activity and conformational stability of the domains of plasma fibronectin

Abstract As measured by two assays of biological activity, fibronectin was readily denatured by heat. Both by the rat liver slice assay and by gelatin-latex agglutination, 90% of the activity disappeared in about 10 min at 60 °C. In contrast, immunological activity, as measured by microcomplement fixation, showed little change at 10 min and was at least 60% as great as unheated fibronectin after 20–50 min at 60 °C. Binding of heparin was unaffected by heating up to 52 min, but at very long times (48 hr at 60 °C), it also was lost. Differential scanning calorimetry of native fibronectin showed three endothermal denaturing transitions, at 68, 82, and 119 °C. Enthalpies of denaturation for the three transitions are approximately 2.6, 0.4, and 0.7 cal/g of flbronectin. These results are consistent with a three-domain structure for fibronectin. The domain which unfolds at 68 °C is associated with gelatin binding and cell. binding. The 82 °C domain appears to be associated with much of the immunological activity, and the 119 °C domain with heparin binding, as well as with some immunological activity. Residual immunological activity after loss of heparin binding may reside in nonordered portions of the molecule.

Survival of native structure and biological activity in fibronectin pasteurized in the presence of sucrose.

Abstract When fibronectin was heated at 60°C in isotonic buffer, all biological activity was lost in less than 1 hr. Structural changes also occurred, as evidenced by the appearance of aggregates on polyacrylamide gels and by a reduction in immunological activity. In contrast, when subjected to the same heat conditions in the presence of 54 to 57% sucrose ( w v ), fibronectin remained virtually in a native state for periods up to 17 hr. After dialysis to remove sucrose, pasteurized fibronectin (10 hr, 60°C) was tested for biological activity by the rat-liver-slice assay, using labeled gelatin-latex particles and by a gelatin-latex agglutination assay. By both methods, sucrose-pasteurized fibronectin exhibited activity nearly as great as unheated controls. When sucrose-pasteurized fibronectin was examined by electrophoresis a single band with no evidence of aggregation was observed. By the method of microcomplement fixation, sucrose-pasteurized fibronectin did not show significant immunological changes compared with unheated fibronectin. When differential scanning calorimetry (DSC) was applied to sucrose-pasteurized fibronectin, there was little or no difference between it and unheated fibronectin in number, temperature, or enthalpy of endothermic transitions. Other substances, which bind to fibronectin (such as heparin and arginine) were shown not to stabilize fibronectin to heat.