Furr H C, Olson J A
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Anal Biochem. 1988 Jun;171(2):360-5. doi: 10.1016/0003-2697(88)90498-8.
Serum retinol (bound to plasma retinol-binding protein, RBP) can be determined by direct injection of as little as 20 microliter of serum or plasma by using size-exclusion high-pressure liquid chromatography (SE-HPLC) with fluorescence detection. Toyo Soda TSK G-3000SW columns (0.75 X 7.5-cm guard column plus 0.75 X 30-cm analytical column) were eluted with 0.2 M NaCl/0.01 M phosphate buffer (pH 6.8) at 1 ml/min, with detection at 280 nm for protein elution. Fluorescence of the retinol-RBP complex was monitored with excitation at 334 nm (interference filter) and emission at 425 nm (long-pass filter). The retinol-RBP complex eluted as two peaks, the holo-RBP-transthyretin complex (apparent molecular weight 70,000) and holo-RBP (apparent molecular weight 9000). Identities of these peaks were established by immunodiffusion assay of the proteins and by extraction and analysis of retinol. Nonideal interactions with the column packing seem to be responsible for the low apparent molecular weight of holo-RBP. The first peak predominated when large volumes of serum (100 to 250 microliters) were injected, and the second when small volumes (5 to 50 microliters) were analyzed. The integrated area of the two fluorescence peaks due to retinol bound to RBP was proportional to the volume of a serum sample injected over the range 5 to 250 microliters.(ABSTRACT TRUNCATED AT 250 WORDS)
血清视黄醇（与血浆视黄醇结合蛋白，即RBP结合）可以通过使用具有荧光检测功能的尺寸排阻高压液相色谱法（SE-HPLC）直接注入低至20微升的血清或血浆来测定。东洋曹达TSK G-3000SW柱（0.75×7.5厘米保护柱加0.75×30厘米分析柱）用0.2M氯化钠/0.01M磷酸盐缓冲液（pH 6.8）以1毫升/分钟的流速洗脱，在280纳米处检测蛋白质洗脱。视黄醇-RBP复合物的荧光通过在334纳米（干涉滤光片）激发和在425纳米（长通滤光片）发射来监测。视黄醇-RBP复合物以两个峰洗脱，即全RBP-甲状腺素转运蛋白复合物（表观分子量70,000）和全RBP（表观分子量9000）。这些峰的身份通过蛋白质的免疫扩散测定以及视黄醇的提取和分析来确定。与柱填充物的非理想相互作用似乎是全RBP表观分子量低的原因。当注入大量血清（100至250微升）时，第一个峰占主导，而分析小体积（5至50微升）时，第二个峰占主导。与RBP结合的视黄醇产生的两个荧光峰的积分面积与在5至250微升范围内注入的血清样品体积成正比。（摘要截短于250字）
