Jones M M, Ogilvie J W, Ackers G K
Biophys Chem. 1976 Sep;5(3):339-50. doi: 10.1016/0301-4622(76)80045-2.
Transport properties of active enzyme species can be studied effectively by layering a small band of enzyme-containing sample on a gel chromatographic column previously saturated with substrate. The column is optically scanned at successive time intervals to yield profiles representing the appearance of chromophoric product or disappearnce of chromophoric substrate. These profiles permit determination of the specific activity and rate of transport of the active species. Initial studies on mechanic of the technique establish the feasibility of accurately determining transport properties of active enzyme species chromatographed on gel columns. Illustrative results are presented for L-glutamate dehydrogenase and for homoserine dehydrogenase studied in both forward and reverse reactions. It is shown that the partititon cross sections derived from the rates of motion of catalytic activity are the same as those determined by equilibrium saturation experiments which directly measure the degree of partitioning by the protein. These results establish the validity of the technique for a variety of future studies. Active enzyme gel chromatography appears comparable in precision to the active enzyme sedimentation technique at current stages of development.
通过在预先用底物饱和的凝胶色谱柱上分层一小条含酶样品，可以有效地研究活性酶物种的传输特性。在连续的时间间隔对柱子进行光学扫描，以产生代表发色产物出现或发色底物消失的图谱。这些图谱可以确定活性物种的比活性和传输速率。对该技术机制的初步研究确定了准确测定在凝胶柱上进行色谱分离的活性酶物种传输特性的可行性。给出了L-谷氨酸脱氢酶和高丝氨酸脱氢酶在正向和反向反应中的说明性结果。结果表明，由催化活性运动速率得出的分配横截面与通过直接测量蛋白质分配程度的平衡饱和实验确定的横截面相同。这些结果确立了该技术在未来各种研究中的有效性。在当前的发展阶段，活性酶凝胶色谱在精度上似乎与活性酶沉降技术相当。
