Protein catalytic elution

An early concern with the HPLC technique was the use of high pressures to achieve high flow rates of the mobile phase through a column packed with microparticulate silica. Recent improvements in column design and operating procedures, however, allow the purification of proteins at modest pressures (e.g., 500 psi) and flow rates (30-60 ml/h). Since it has been reported that C 3-alkyl chains are compatible with catalytic activity of adsorbed and eluted proteins, but larger alkyl substituents may cause denaturation (26), the use of reversed-phase columns of medium polarity, e.g., —C Hy-phenyl, when combined with a judicious choice of organic modifier and salt concentrations (e.g., isopropanol and phosphate) at pH... 

The 7-0-hemlsucclnate-7-desacetyl derivative of forskolin has been coupled to Sepharose to form an affinity resin. 2 Solubilized adenylate cyclase from rat brain and heart, but not pigeon erythrocytes, binds to this resin and can be eluted from the resin when forskolin is included in the buffer. Preparations of adenylate cyclase devoid of Ns activity are recovered from this column consistent with the proposal that forskolin binds to the catalytic subunit(s). Adenylate cyclase preactivated with guanosine-5, 3>Y i idodiphosphate (GppNHp) also binds to the forskolln-Sepharose column and elutes from the column as a complex of the catalytic subunlt(s) and the activated Ns protein. 

This conception works out as shown in Scheme 10 CTP 23 formed by the above described sequence is directly consumed by -acetyl neuraminic acid 26 under the catalytic influence of cytidine-5 -monophosphosialate synthase (E.C. 2.7.7.43). This enzyme is isolated from calf brain by ammonium sulfate precipitation (2 5) and subsequent affinity chromatography. The stationary phase consists of CNBr-activated Sepharose 4B reacted with p-[3-(2-amino ethylthio)propyl]-iV-acetyl neuraminic acid 27, which is synthesized by radiating a mixture of the allyl glycoside and cysteamine to achieve radical C-S bond formation (24), The behavior of methyl p-N-acetyl-neuraminic acid as an inhibitor is in accordance with Zbiral s findings (25), where the methyl a-glycoside has been shown to compete with the native substrate for the enzyme, and thus 27 is recommended to be an ideally suited ligand (Scheme 9). A typical analytical run is shown in Scheme 9. Due to elution of the protein fraction by a salt gradient, the transfer to a preparative scale is rather difEcult denaturation occurs and thus a drop in activity down to 6% is observed. 

---