Keiser H D, Adlersberg J B, Steinman H M
Biochem J. 1982 Jun 1;203(3):683-9. doi: 10.1042/bj2030683.
Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.
从牛鼻软骨的4M - 氯化胍提取物的胰蛋白酶消化物中制备出相对均一的蛋白聚糖片段级分。通过离子交换色谱法将含糖胺聚糖的片段与非蛋白聚糖污染物分离，并在解离条件下通过平衡密度梯度离心进行分级分离。将最高浮力密度的级分在琼脂糖4B柱上进行色谱分离，用软骨素酶ABC消化后再在琼脂糖6B柱上进行色谱分离，得到两个不同的级分：级分B/6B - 4包含来自蛋白聚糖单体硫酸软骨素区域的片段，级分B/6B - 2包含来自富含硫酸角质素区域的片段，很可能还包括一个含硫酸软骨素的单体片段。通过丹磺酰氯分析，级分B/6B - 2以丙氨酸和亮氨酸作为唯一的N端氨基酸，级分B/6B - 4以异亮氨酸和亮氨酸作为占主导地位的N端氨基酸，这表明这些软骨蛋白聚糖单体片段制剂具有相对均一性。
