Journal of Chromatography , 368 (1986) 283-289 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROM. 18 866 COMPARISON OF REVERSED-PHASE COLUMN MATERIALS FOR HIGH- PERFORMANCE LIQUID CHROMATOGRAPHY OF PROTEINS RUURD VAN DER ZEE*, THEA HOEKZEMA, SYTSKE WELLING-WESTER and GJALT W. WELLING Laboratorium voor Medische M icrobiologic, Rijksuniversiteit Groningen, Oostersingel 59, 9713 EZ Groningen (The Netherlands) (Received June 9th, 1986) SUMMARY Nine reversed-phase materials with various bonded phases from different sup- pliers were studied for the separation of hydrophilic proteins with two solvent sys- tems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems. INTRODUCTION In the past five years several high-performance liquid chromatography (HPLC) modes (ion-exchange, size-exclusion, reversed-phase, hydrophobic interaction) have become important for the purification of proteinsl. However, adaptation of these techniques to purification schemes of integral membrane proteins proceeds slowly since the methodology to handle these proteins is still in its infancy. Membrane pro- teins show a marked tendency to aggregate, which is not surprising in view of their natural environment, which is a bilayer of lipid molecules. In our studies for the development of HPLC methods for the purification of integral membrane proteins, Sendai virus membrane proteins are used as model pro- teins. Sendai virus, a paramyxovirus of mice, contains three proteins that are as- sociated with the lipid bilayer membrane, the matrix protein M (Mr = 38 000), the hemagglutinin-neuraminidase protein HN (MT = 67 000) and the fusion protein F. The,latter consists of two subunits, Fr (M, = 50 000) and zyxwvutsrqponmlkjihgfedcbaZ F2 (Mr = 14 000), con- nected by disulphide bonds. These proteins can be selectively extracted from purified virions with Triton X-100 in the presence of 1 M salt2. Previous studies have shown that Sendai virus membrane proteins can be purified by reversed-phase HPLC (RP-HPLC)3+4. However, the mass recovery of these proteins is generally poor. 0021-9673/86/$03.50 0 1986 Elsevier Science Publishers B.V.