The Development of Novel Phase Separations

In Chapter 2, we summarized the myriad of phase separations that that facilitate ligand binding analyses (Winzor et al., 1995). At low concentrations of acceptor, a phase separation’s ability to reduce non-specific free ligand adsorption is a major performance limiting factor. In Chapter Three, we presented the development of the RevMAPP protocol which allowed us to directly probe nucleotide binding to low nanomolar concentrations of RNAP during transcription elongation. However, we designed the RevMAPP protocol specifically for transcription, and its application may be limited to the analyses of ligand- acceptor pairs compatible with biotin affinity purification. In addition to RevMAPP, we pursued the invention of several novel phase separation procedures to remove nucleotides (a charged ligand) from a solution containing acceptor. Our methods were designed to

maximize the speed of NTP removal and reduce non-specific ligand adsorption while capturing protein acceptors for binding analysis. To develop new, versatile, high

performance separations for ligand acceptor analyses, we modified or combined the concepts of existing dialysis (Svensson, 1946; Colowick et al., 1969; Ford et al., 1984), filter binding (Harris et al., 1988) and electrophoresis techniques (Takeo et al., 1972; Garner et al., 1981).

4.1.1 Small Volume Electrodialysis

The speed of dialysis techniques are limited by the mass transport of ligands to a semi permeable membrane. In traditional dialysis, stirring is used to eliminate solution

concentration gradients and increase mass transport of ligands to the dialysis membrane. Since NTPs are charged ligands, we were curious to see if a negative voltage would drive nucleotides to the membrane faster than diffusion alone. By decreasing the sample solution volume (α-phase), we hoped to reduce the time require for the phase separation by reducing the distance molecules needed to travel. The idea of a small sample volume was also attractive to for the purposed of material (acceptor protein) conservation. Here we present the development and performance of small volume electrodialysis (SVED).

4.1.2 Reverse Microfiber Dialysis

Microfibers have been used as a versatile sample collection tool for drug-protein binding analyses and the detection of neurotransmitters and amino acids both in vivo and in vitro (Oravcová et al., 1996; Liu et al., 1999; Zhou et al., 2004). Microfiber use for

traditional equilibrium dialysis has been quantitatively compared to filter binding for

pharmacological studies (Herrera et al., 1990). However, microdialysis is typically used for sample collection. Microfiber sample collection has been used in-line with analysis by HPLC (Wang et al., 1997) and capillary electrophoresis (Hogan et al., 1994). Recently, microfibers were used to de-salt samples in-line with nanoelectrospray ionization-mass spectrometry; the authors infused the analyte into the fiber, rather than using the microfiber as a sample

collection vehicle (Jakubowski et al., 2005). We wanted to examine the possibility of purifying ligand-protein mixtures with microfibers by infusing the fiber with the sample

solution instead of collecting free ligand from the exterior of the microfiber. Rather than capture a sample of the free ligand, in our case the perfusate of the microfiber would contain only the bound complex and free acceptor, assuming the system was allowed to reach dialysis equilibrium with a large surrounding β-phase. In dialysis, sources of non-specific NTP adsorption include any experimental equipment that confine the dialysis α-phase solution. We postulated that sources of non-specific adsorption could be reduced by using microfibers made from regenerated cellulose. The microfiber membrane would completely house the dialysis solution, reducing NTP solution contact with experimental materials. Plus, microfibers have a very high membrane area to volume ratio. Compared to conventional dialysis apparatuses, the geometry of microfibers would greatly decrease diffusion limited dialysis equilibration time. We felt that the very small inner volume (5 µL) of microfibers would also aid in the conservation of protein acceptor.

Microfibers have been previously used as filtering vehicles. One study using hollow microdialysis fibers to purify the contents of a PCR reaction for analysis by esi-mass

spectrometry reported that Mg+2 was readily removed from the interior of a hollow

microdialysis fiber (MWCO = 13,000) at a sample infusion flow rate of 2µL·min-1 (Hannis et al., 1999). However, only 20 to 30 % of dNTPs were removed from solution. The authors attribute dNTP retention to the “large solvation sphere associated with the triphosphate groups,” and also cite “base stacking of monophosphate nucleotides.” They claim the large solvation sphere and the base stacking increased the “apparent molecular weight” of dNTPs. We were not at all convinced that dNTP solvent dynamics or base stacking would make a small nucleotide (MW < 1000 Da) act as if it were larger than 13,000 Daltons. Non-specific adsorption of dNTP to the regenerated cellulose walls may have partially caused the retention

of nucleotide. It is also possible, but unlikely, that the solution did not fully reach dialysis equilibrium due to Gibbs-Donnan effects. A third possibility is that the complex PCR mixture containing DNA polymerase, primers, PCR products, etc, simply clogged the pores of the fiber. The findings by Hannis et. al. did not discourage us from pursuing reverse microfiber dialysis.

4.1.3 Short Travel Gel Electrophoresis

A variety of agarose and polyacrylamide electrophoretic techniques are commonly used to separate biological molecules with very low background, especially when using radiochemical methods. Electrophoresis has been used for nearly 30 years to ascertain binding parameters for DNA-protein interactions (Garner et al., 1981). However, the utility of using an electrophoresis gel as a simple filter for ligand-acceptor mixtures has never been investigated. The electrophoresis of NTPs through gel materials is considerably faster than the migration of large proteins. We theorized that application of a voltage to a short plug of agarose or polyacrylamide might quickly pass NTPs and in the time required to for proteins to enter the gel matrix. Our invention of short travel gel electrophoresis (STaGE) for equilibrium binding assays is described below.