Characterization of the soluble proteins from rabbit eye lens.
Abstract Fractionation of the soluble proteins of rabbit lens by gel chromatography on Sepharose 6B revealed six components, viz. HM-, α-, β 1 -, β 2 -, β 3 - and γ-crystallin. On isoelectric focusing α-crystallins revealed a diffuse region, whilst approximately 10 bands were found for the β-crystallins and 7–8 components for γ-crystallin. Immunoelectrophoretically HM-crystallin revealed two precipitin lines, one in the α- and one in the β-mobility range. Sedimentation coefficients of 18·0, 8·0, 3·7, 2·2 and 2·1 S were calculated for α-, β 1 -, β 2 -, β 3 - and γ-crystallin, respectively. By isoelectric focusing in the presence of 6 m -urea, 13 bands between pH 5·3 and 6·9 were found for HM-crystallin, approximately six bands between pH 5·6 and 6·9 for α-crystallin, about 13 bands between pH 5·9 and 7·0 for the β-crystallins and 7–8 major bands between pH 7·0 and 7·8 for γ-crystallin. Molecular weight estimation by SDS gel electrophoresis revealed values between 16 000 and 39 000 for HM-crystallin and the β-crystallins, whereas values of approximately 21 000 and 20 500 were found for α- and γ-crystallin.
High-performance gel permeation chromatography of bovine eye lens proteins in combination with low-angle laser light scattering. Superior resolution of the oligomeric beta-crystallins.
Calf lens extracts were subjected to high-performance gel permeation chromatography on TSK GEL G4000 SW and G3000 SW columns (fractionation range: 5 x 10(6)-10(4) daltons) and resolved into thirteen crystallin fractions: HM-, alpha-, six beta H-, two beta L-, beta S- and two gamma-crystallins. Molecular weights were determined using a low-angle laser light scattering detection system. The weight average and number average molecular weights for cortical alpha-crystallin, 860,000 and 740,000, respectively, reveal a polydispersity factor of 1.16 for this heterogeneous protein. The eight different beta-crystallin fractions could be found with practically all possible oligomeric structures from dimers to aggregates larger than dodecamers. Different structures are found for the predominant beta H-crystallin fractions, viz., hexamers and pentamers, in the extracts from cortex and nucleus. Additional identification of the fractions by sodium dodecyl sulphate gel electrophoresis and isoelectric focusing in the presence of urea also indicated that semi-preparative application of this high-performance technique is possible. The co-elution of putative cytoskeletal proteins with some beta-crystallins was remarkable; moreover, co-elution of FM-crystallin with beta S-crystallin is discussed. A 23,000-dalton fraction, mainly found in the cortical region, most likely corresponds to the 24,000-dalton gamma-crystallin preparation obtained from cattle lens cortices. It is questioned whether the similarities between this fraction and beta S-crystallin are merely coincidental.
Control of reflective cracking
The first is 'fatigue' cracking of the layer. This type of cracking is the result of the application of loads on the pavement and is controlled by appropriate thickness design which 'ill not be discussed in detail here. Thickness design includes not only the design strength and thickness of the stabilised, but other design elements such as delaying reflection cracking, or limiting stresses in the stabilised layer, by adopting a minimum thickness of overlying material. This may include an unbound layer utilising the 'upside down' pavement approach. The actual thickness required of the overlying material varies but for example PIARC (Ref.1) recently reported when the thickness of coated materials exceeds 20 cm the emergence of shrinkage cracks at the surface is generally uncommon; when it is 15 cm thick, the cracks emerge at the surface, but do not shorten the life of the surface course, especially if they are sealed. This experience has been gained 'with legal axle loads of 130 kN, considerably greater than the Australian legal axle load.