Li YZhang.Y.and Yan,H.(196)Biol Chem 1,3-804 GMP titration into guanylate kinase 191 1.90 ◇ 0.mM GMP 1.88 187 Repeat for multiple crosspeaks 1.85 184 1.83 010203040 50 GMP(ul) A+B K0之AB Ko=KouKon 上eagmeoeeenteeneon6m Lower limit for K1uM 8eaeaeghieoalshsaeoeogeerndsoweeangessw *This is a big assum especially if there are any significant conformational rearrangements involved. .g.Emerson et al(19)Biochemistry 346911-6918 Li, Y., Zhang, Y., and Yan, H. (1996) J. Biol. Chem. 271, 28038–28044 GMP titration into guanylate kinase 0.58mM GK Adding 13.3 mM GMP 29 µM KD Weak binding Fast exchange Repeat for multiple crosspeaks e.g. Emerson et al (1996) Biochemistry 34 6911-6918 Look for the largest chemical shift difference between the free and the bound forms that still shows fast exchange e.g. it is 100 Hz Koff > 100 sec-1 Kon is diffusion limited: 108 M-1 sec-1 * Lower limit for KD ~ 1 µM So if the free and bound chemical shifts are close together and slow exchange is still observed, the KD must be low * This is a big assumption, especially if there are any significant conformational rearrangements involved. A + B AB kon koff KD = Koff/Kon