Garnett J, Dieppe P
Department of Medicine, Bristol Royal Infirmary, U.K.
Biochem J. 1990 Mar 15;266(3):863-8.
The effects of potential serum inhibitors upon the growth of calcium hydroxyapatite (HAP) crystals were studied in vivo using a pH-stat system. Whole serum caused a marked decrease in crystal growth in a dose-dependent manner. At a protein concentration of 13 micrograms/ml, whole serum reduced the initial rate of crystal growth from 84 mumol of KOH/h to 48 mumol of KOH/h. Serum components were separated by ultrafiltration (10,000 Da cut-off). The high-molecular-mass fraction containing serum proteins gave an initial rate of crystal growth of 48 mumol of KOH/h compared with 64 mumol of KOH/h given by the low-molecular-mass components. Thus, two-thirds of the inhibitory activity was associated with proteins and other serum macromolecules, whilst the remainder of the activity was associated with the low-molecular-mass components. Albumin-depleted serum showed an initial rate of crystal growth of 59 mumol of KOH/h, whilst albumin purified by affinity chromatography gave an initial rate of crystal growth of 56 mumol of KOH/h at the same protein concentration. Albumin, therefore, not only accounts for half of the protein concentration in serum, but also contributes half of the inhibitory activity of the high-molecular-mass fraction. Heat denaturation of albumin dramatically enhanced the inhibition of HAP seeded growth with the initial rate of crystal growth falling to 27 mumol of KOH/h after treatment compared with 62 mumol of KOH/h before denaturation. Isoelectric focusing indicated that the tertiary and secondary structure, and hence the distribution of surface charge of albumin, are altered by heat denaturation. Gels showed a mixture of species with isoelectric points ranging from 6.0 to 5.0 compared with the native protein value of 4.7. These data suggest that adsorption of serum proteins to the growing HAP crystals is one mechanism of growth inhibition. It is also clear that the most abundant serum protein, albumin, is an important mediator of this process.
利用pH计系统在体内研究了潜在血清抑制剂对羟基磷灰石（HAP）晶体生长的影响。全血清以剂量依赖的方式显著降低晶体生长。在蛋白质浓度为13微克/毫升时，全血清将晶体生长的初始速率从84微摩尔氢氧化钾/小时降至48微摩尔氢氧化钾/小时。通过超滤（截留分子量10,000道尔顿）分离血清成分。含有血清蛋白的高分子量部分晶体生长的初始速率为48微摩尔氢氧化钾/小时，而低分子量成分的初始速率为64微摩尔氢氧化钾/小时。因此，三分之二的抑制活性与蛋白质和其他血清大分子相关，而其余活性与低分子量成分相关。去除白蛋白的血清晶体生长的初始速率为59微摩尔氢氧化钾/小时，而通过亲和色谱纯化的白蛋白在相同蛋白质浓度下晶体生长的初始速率为56微摩尔氢氧化钾/小时。因此，白蛋白不仅占血清蛋白质浓度的一半，而且还贡献了高分子量部分抑制活性的一半。白蛋白的热变性显著增强了对HAP接种生长的抑制作用，处理后晶体生长的初始速率从变性前的62微摩尔氢氧化钾/小时降至27微摩尔氢氧化钾/小时。等电聚焦表明，热变性改变了白蛋白的三级和二级结构，从而改变了其表面电荷分布。凝胶显示出等电点范围从6.0到5.0的多种物种混合物，而天然蛋白质的等电点为4.7。这些数据表明血清蛋白吸附到生长的HAP晶体上是生长抑制的一种机制。同样明显的是，血清中最丰富的蛋白质白蛋白是这一过程的重要介质。
