Electric birefringence studies of cartilage proteoglycan aggregation.
Electric birefringence studies of cartilage proteoglycan aggregation.
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软骨蛋白多糖聚集的电双折射研究。
DOI：
10.1002/bip.1977.360160617
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发表时间：
1977
期刊：
影响因子：
2.9
通讯作者：
H. Muir
中科院分区：
文献类型：
--
作者：
A. R. Fowermaker;M. Isles;B. Jennings;T. Hardingham;H. Muir
在过去的十年中，人们对使用电光方法表征稀溶液中的大分子越来越感兴趣。要利用的最突出的效应是电双折射或克尔效应。对于生物聚合物溶液，其根源在于当溶液受到施加的电场时溶质分子的取向。 2.3 使用脉冲电场是有利的。然后，分子溶液中引起的伴随双折射本质上变成瞬态，其变化速率指示了溶剂环境内分子取向的速率。通常通过分析瞬态双折射的衰减率来直接测量分子旋转弛豫时间 (7)。由于该参数大约是致密分子最大尺寸的三次方的函数4，因此它是分子构象变化和缔合的极其敏感的指标。在本次交流中，我们通过一些电双折射测量提请人们注意该方法在生物物理学和医学物理领域的潜力，我们相信，这些测量可以快速确认最近提出的软骨蛋白聚糖与透明质酸链复合的模型。软骨蛋白聚糖由约 400 nm 长的蛋白质主链和约 100 个侧链组成，每个侧链长约 40 nm，主要由硫酸软骨素组成 [图 1]。啦）]。硫酸角质素还会出现额外的较短侧链。蛋白多糖存在于软骨中，主要是含有透明质酸的大聚集体。根据凝胶色谱、粘度和电子显微镜数据，认为聚集体由延伸的透明质酸链组成，蛋白多糖以放射状方式规则地附着在该透明质酸链上（每 10,000 分子量单位最多有一个）[图 1]。 lc）]。每个蛋白多糖分子都有一个球状蛋白质头，它连接着透明质酸分子。聚集体的确切生物学作用尚不清楚。然而，人们注意到，在严重纤维化的人类软骨中，聚集体的量明显少于正常、健康的软骨结缔组织。 8 对猪喉蛋白多糖样品进行电双折射测量，该样品按照其他地方概述的方式制备。将高达 600 V cm-' 的电场和长达 160 毫秒的脉冲持续时间施加到每毫升含 209 pg 糖醛酸（约 0.8 mg ml-' 蛋白多糖）的蒸馏水中的蛋白多糖溶液。图1b)的光学响应表明样品的大双折射和3.6(k0.3)毫秒的旋转弛豫时间。以0.1ml至10ml蛋白聚糖溶液的步骤添加每ml 20pg糖醛酸（约60pgml-'透明质酸）的透明质酸溶液，并记录连续的光学瞬变。在图1d)中，我们呈现了透明质酸有效饱和后稳定状态的迹线。有两个观察结果立即显而易见。首先，双折射幅度发生了显着变化并且可能改变了符号。尽管在这项初步研究中没有考虑到光散射的影响，但双折射符号的变化与从蛋白多糖到聚集分子的光学各向异性的逆转是一致的。与蛋白多糖分子的主要尺寸相关的折射率成为聚集体横轴的折射率。其次，复合物的弛豫时间约为 650 (f30) 毫秒，约为组成蛋白聚糖分子的 180 倍。
During the past ten years there has been an increasing interest in the use of electro-optical methods for the characterization of macromolecules in dilute solution. The most prominent effect to be utilized is that of electric birefringence or the Kerr effect.'With biopolymer solutions, this has its origin in the orientation of the solute molecules as the solution is subjected to an applied electric field. 2.3 It is advantageous to use pulsed electric fields. Then, the accompanying birefringence induced in the molecular solution becomes transient in nature, with the rates of change an indication of the rates of molecular orientation within the solvent environment. The decay rate of the transient birefringence is usually analyzed to give a direct measure of the molecular rotary relaxation time (7). As this parameter is approximately a function of the third power of the greatest dimension of compact molecules, 4 it is an extremely sensitive indicator of molecular conformation changes and associations. In this communication we draw attention to the potential of the method in the field of biophysics and medical physics through some electric birefringence measurements which, we believe, provide rapid confirmation of a model recently proposed5 for the complexing of cartilage proteoglycans to hyaluronic acid chains. Cartilage proteoglycans consist of a protein backbone about 400 nm long, with approximately 100 sidechains, each about 40 nm in length, of predominantly chondroitin sulphate [Fig. la)]. Additional shorter side chains of keratan sulphate also occur. The proteoglycans are found in cartilage, mainly as large aggregates involving hyaluronic acid. From gel chromatographic, visc~ simetric,~ and electron microscopic6 data the aggregate is thought to consist of an extended hyaluronic acid chain to which the proteoglycans are regularly attached (up to one per 10,000 molecular weight unit) in a radial manner [Fig. lc)]. Each proteoglycan molecule has a globular protein head which hinds to the hyaluronic acid m~ lecule.~ The exact biological role of the aggregate in unknown. However, it has been noted that in severely fibrillated human cartilage, the amount of aggregate is markedly less than in regular, healthy cartilage connective tissue. 8Electric birefringence measurements were made on a sample of pig laryngeal proteoglycan, prepared in the manner outlined el~ ewhere.~ Electric fields of up to 600 V cm-'and for pulse durations of up to 160 msec were applied to solutions of proteoglycans in distilled water containing 209 pg uronic acid per ml (approx 0.8 mg ml-'proteoglycan). The optical response of Figure lb) indicates the large birefringence of the sample and a rotary relaxation time of 3.6 (k0. 3) msec. A hyaluronic acid solution of 20 pg uronic acid per ml (approximately 60 pg ml-'of hyaluronic acid) was added in steps of 0.1 ml to 10 ml of the proteoglycans solution and successive optical transients were recorded. In Figure Id) we present a trace for the stable condition after effective saturation with hyaluronic acid. Two observations are immediately apparent. First, the birefringence amplitude has changed significantly and has possibly changed sign. Although the effect of light scattering has not been allowed for in this initial study, a change in birefringence sign is consistent with the reversal of the optical anisotropy from the proteoglycan to the aggregated molecules. The refractive index associated with the major dimension of the proteoglycan molecule becomes that of the transverse axes of the aggregate. Second, the relaxation time of the complex is of the order of 650 (f30) msec, some 180 times that of the constituent proteoglycan molecules.