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  • The ultrafiltration approach has been used to


    The ultrafiltration approach has been used to measure 5-Formyl-UTP equilibrium BIEs for a number of enzyme systems, including 5-Formyl-UTP hexokinase (Lewis and Schramm, 2003a, Lewis and Schramm, 2003b), thymidine phosphorylase (Birck & Schramm, 2004), purine nucleoside phosphorylase (Murkin, Tyler, & Schramm, 2008), orotate phosphoribosyltransferase (Zhang & Schramm, 2011), and uridine phosphorylase (Silva, Kipp, & Schramm, 2012). Recently, the ultrafiltration method for BIE measurements was employed by Klinman and coworkers in the study of ES interactions for catechol O-methyltransferase (Zhang, Kulik, Martinez, & Klinman, 2015) and glycine N-methyltransferase (Zhang & Klinman, 2016). Technical application of the ultrafiltration approach to BIEs is detailed in these publications and has also been detailed for direct ligand binding studies (Schramm, 1976). Other methods that have been implemented to partition free and bound substrate fractions for BIE measurements include the use of protein concentrator (Griswold, Castro, Fisher, & Toney, 2012) and G-25 size-exclusion spin tubes (Linscott et al., 2016). In the approach using concentrator tubes, partitioning of the ES complex and free substrate occurs based on passage through a semipermeable membrane. However, in contrast to both the equilibrium dialysis and ultrafiltration methods, the solution of free substrate that passes through the membrane is removed from the inferior aspect of the system by centrifugation, which prevents the equilibration of free substrate back across the membrane after initial diffusion. Partitioning of the unbound and bound substrate fractions by G-25 spin columns occurs due to retention of the free small-molecule substrate on the resin and early elution of the ES complex. This approach leads to complete separation of the ES complex and free substrate fraction, which could disrupt the initial equilibrium state of the system, particularly for weakly binding substrates. Recently, our lab reported BIEs on substrate binding for dihydropteroate synthase (Stratton, Namanja-Magliano, Cameron, & Schramm, 2015) and the human protein lysine N-methyltransferase NSD2 (Poulin, Schneck, Matico, Hou, et al., 2016) using a commercially available rapid equilibrium dialysis (RED) device (Thermo Fisher Scientific). In this chapter, we describe an equilibrium dialysis approach for measuring BIEs using the RED device as implemented in our study on the effects of nucleosome binding on the substrate bonding environment of NSD2.
    Materials The buffer mixtures and substrates used to measure BIEs on the association of S-adenosyl-l-methionine (SAM) and NSD2 in binary and ternary complexes are summarized in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7.
    Measurement of Equilibrium BIEs Using the RED Device The RED device (Thermo Fisher Scientific) consists of a polypropylene base with a standard 96-well plate footprint that holds 48 dialysis inserts. Each dialysis insert is constructed as two wells separated by a vertical cylinder of cellulose membrane. The volume contained within the cylinder of dialysis membrane will be referred to as the “sample well,” and the outer volume surrounding the dialysis membrane will be referred to as the “buffer well” (Fig. 3). In the BIE experiment with SAM and NSD2, unbound substrate (i.e., [3H]SAM and [14C]SAM) can freely diffuse across the 8-kDa cutoff membrane, whereas the enzyme and ES complex (i.e., bound substrate) cannot. Following equilibration of the substrate across the dialysis membrane, the sample well contains counts per minute (cpm) corresponding to both the bound (3Hswbound; 14Cswbound) and unbound (3Hswunbound; 14Cswunbound) substrate in a total volume Vsw. By contrast, the buffer well contains cpm for only the unbound substrate (3Hbw; 14Cbw) in a total volume Vbw. Accordingly, at equilibrium, the cpm per unit volume for the unbound substrate in the sample well is equivalent to that of the buffer well: