Cheung D T, Benya P D, Hofer D P, Nimni M E
Coll Relat Res. 1981 Apr;1(3):247-56. doi: 10.1016/s0174-173x(81)80002-7.
CNBr peptides from insoluble bovine cortical bone collagen were analyzed using a 2-D mapping technique. The major type 1 collagen CNBr peptides were detected by fluorography after general-labelling with 3H-NaBH4 in dimethyl-formamide. These maps were similar to those visualized by coomassie blue staining and demonstrated a proportional decrease of alpha 1CB6. New groups of peptides, different from those normally present in soluble type I collagen were detected. Some of these peptides were slightly larger and more acidic than alpha 1CB6 and were highly labelled when the demineralized bone was specifically labelled for the presence of aldehydes and crosslinks with 3H-NaBH4 in a phosphate buffer, pH 7.4. Based on the size and charge characteristics of these specifically labelled peptides, they were tentatively identified as crosslinking peptides containing different combinations of alpha 1CB6, alpha 1CB0,1 and alpha 1CB5. The specificity of the labelling method using 3H-NaBH4 in phosphate buffer was demonstrated by the detection of other known crosslinked peptides and by the virtual absence of label in alpha 1CB7, CB8, and CB3. We feel that this simple methodological approach developed in these experiments will prove to be very useful in the analysis of collagen crosslinks present in insoluble collagens derived from normal tissues of various ages as well as from pathological states.
采用二维图谱技术分析了不溶性牛皮质骨胶原蛋白的溴化氰肽段。在用3H-NaBH4在二甲基甲酰胺中进行通用标记后，通过荧光自显影检测主要的I型胶原蛋白溴化氰肽段。这些图谱与考马斯亮蓝染色显示的图谱相似，并表明α1CB6呈比例下降。检测到了与可溶性I型胶原蛋白中通常存在的肽段不同的新肽段组。其中一些肽段比α1CB6略大且酸性更强，当脱矿骨在pH 7.4的磷酸盐缓冲液中用3H-NaBH4特异性标记醛和交联键的存在时，它们被高度标记。根据这些特异性标记肽段的大小和电荷特征，初步将它们鉴定为含有α1CB6、α1CB0,1和α1CB5不同组合的交联肽段。通过检测其他已知的交联肽段以及α1CB7、CB8和CB3中几乎没有标记，证明了在磷酸盐缓冲液中使用3H-NaBH4的标记方法的特异性。我们认为，在这些实验中开发的这种简单方法在分析来自不同年龄正常组织以及病理状态的不溶性胶原蛋白中存在的胶原交联方面将被证明非常有用。
