Selective Removal of Ribonucleases from Solution with Covalently Anchored Macromolecular Inhibitor
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- 作者：M. Habibur Rahman ; Insug Kang ; Raymond G. Waterbury ; Upvan Narang ; Frank V. Bright ; and Jui H. Wang
- 刊名：Analytical Chemistry
- 出版年：1996
- 出版时间：January 1, 1996
- 年：1996
- 卷：68
- 期：1
- 页码：134 - 138
- 全文大小：302K
- 年卷期：v.68,no.1(January 1, 1996)
- ISSN：1520-6882
文摘
Poly[2'-O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] hasbeen found to be a potent inhibitor in solution for RNasesA, B, S, T1, T2, and H as well asphosphodiesterases Iand II. Kinetic measurements with RNase B and RNaseT1 showed DNP-poly(A) to be a reversiblecompetitiveinhibitor with KI equal to 1.03 and 1.05 
M,respectively.Data on the quenching of fluorescence of RNase T1byDNP-poly(A) indicate the existence of more than oneRNase-binding site in each DNP-poly(A) molecule.Byattaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column wasprepared for selective removal of RNases from aqueoussolutions by simple filtration. It was found that a1000-fold reduction in RNase concentration can be obtainedby passing either 7.0 M or 7.0 nM RNase A solutionthrough a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solutionand subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedlywith no significant decrease in RNase-binding affinity andcapacity. By titration of the derivatized beads withRNase,the first dissociation constant (Kd) and bindingcapacityfor the bound enzyme can be determined. TheKd wasfound to be 0.66 M for RNase B and 0.48 M for RNaseT1; the corresponding binding capacities were foundtobe 21.0 × 10-8 and 9.6 ×10-8 mol/g, respectively.
M,respectively.Data on the quenching of fluorescence of RNase T1byDNP-poly(A) indicate the existence of more than oneRNase-binding site in each DNP-poly(A) molecule.Byattaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column wasprepared for selective removal of RNases from aqueoussolutions by simple filtration. It was found that a1000-fold reduction in RNase concentration can be obtainedby passing either 7.0 M or 7.0 nM RNase A solutionthrough a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solutionand subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedlywith no significant decrease in RNase-binding affinity andcapacity. By titration of the derivatized beads withRNase,the first dissociation constant (Kd) and bindingcapacityfor the bound enzyme can be determined. TheKd wasfound to be 0.66 M for RNase B and 0.48 M for RNaseT1; the corresponding binding capacities were foundtobe 21.0 × 10-8 and 9.6 ×10-8 mol/g, respectively.