High-performance liquid chromatography of Sendai virus membrane proteins

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ming some well known MCMs in the CLAS program was to obtain an optimal integration of the MCM and the leave-one-out procedure that is used for evaluationl. With most of the currently available programs, the only way to obtain a leave-one-out evaluation is to perform it literally, which requires as many runs of the computer program as there are objects in the data set. Clearly this is not acceptable in practical situations. CLAS is able to perform leaveone-out evaluations with statistical linear discriminant analysis (SLDA), ALLOC3, SIMCA4, CLASSY5*6(implemented here for the first time), and also multiple linear regression. An advantage of the SIMCA reimplementation is the computation of probabilities. However, it should be noted, that not all options of other implementations of these methods are present in CLAS. Other features of CLAS include a new technique for missing data handling7, and the possibility of using Horn’s test to find the number of principal components to be used in the SIMCA or CLASSY class models’. Table I shows some technical details of CLAS and Table II gives the procedures that are currently implemented with their major functions. CLAS is available from the authors as pre-release, fit for a Control Data Cyber mainframe, under the NOS-

(-BE) operating system, using Pascal-6000. However, at this moment it is being adapted for use on a IBM microcomputer.

References H. van der Voet, J. B. Hemel, P. M. J. Coenegracht,

Anal.

Chim. Acta, (1986) in press.

J. B. Hemel and H. van der Voet, Anal. Chim. Actu, (1986) in press. D. Coomans, D. L. Massart, I. Broeckaert and A. Tassin, Anal. Chim. Actu, 133 (1981) 215. S. Wold and M. Sjastrbm, in B. R. Kowalski (Editor), Chemometrics: Theory and Application (ACS Symp. Ser. 52),

American Chemical Society, Washington, DC, 1977, p. 241. H. van der Voet and D. A. Doornbos, Anal. Chim. Actu, 161 (1984) 115. H. van der Voet, J. B. Hemel and P. M. J. Coenegracht, Anal. Chim. Actu, (1986) in press. J. B. Hemel, H. van der Voet, F. R. Hindriks and W. van der Slik, Anal. Chim. Actu, in press. P. E. Green, Analyzing Multivuriute Data, the Dryden Press, Hinsdale, 1978, p. 366.

Jan B. Hemel is at the Central Laboratory for Clinical Chemistry, University Hospital Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands. Hilko van der Voet is at the Research Group Chemometrics, Pharmaceutical Labs., State University of Groningen, A. Deusingluun 2, 9713 A W Gronigen, The Netherlands.

biotechnology focus

High-performance liquid chromatography of Sendai virus membrane proteins Gjalt W. Welling, Ruurd van der Zee and Sytske Welling-Wester Groningen, The Netherlands The three membrane proteins of Sendai virus can be extractedfrom the membrane by non-ionic detergents. The extracted proteins serve as a model mixture for the development of high-performance liquid chromatographic methods for the purification of hydrophobic membrane proteins ,

Introduction

The rapidly evolving methods of biotechnology which allow production of viral membrane proteins that were hitherto difficult to obtain in sufficient 0165-9936/86/$02.00.

quantities, necessitate adequate chromatographic purification methods. Integral membrane proteins are tightly associated with a bilayer of phospholipid molecules. A significant part of the integral membrane proteins, generally one or more membranespanning helices mainly consisting of hydrophobic amino acids, interacts with the hydrophobic tails of the lipid molecules. Integral membrane proteins are generally more hydrophobic than an average protein and their purification requires methodologies which take advantage of their hydrophobic properties. We have chosen the integral membrane proteins of Sendai virus as model proteins to study various methods for the high-performance liquid chromatographic (HPLC) purification of integral membrane proteins. 0

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NP I M

Fig. I. Schematic representation of Sendai virus. The various structural components except for the L protein are indicated.

Sendai virus Sendai virus is classified as a member of the family of paramyxoviridae. The term ‘myxo’ (from the Greek myxa, meaning mucus) reflects the fact that paramyxoviruses (and also orthomyxoviruses) have special affinity for mucopolysaccharides and glycoproteins. Influenza viruses A and B are representatives of the family of orthomyxoviruses. Both groups exhibit similar properties, such as agglutination of erythrocytes and the presence of neuraminidase in some members of the group, and can be relatively easily cultivated in chicken embryos. The family of paramyxoviridae comprises three genera’, morbillivirus, pneumovirus and the genus of paramyxoviruses, which includes Sendai virus of mice, Simian virus 5 (SV5), four types of human parainfhrenza viruses, mumps virus and Newcastle disease virus (NDV) 6f birds. Paramyxoviruses are enveloped viruses, 150-250 nm in diameter, but larger virus particles are quite common and filamentous forms up to a length of several micrometers have been described. The Sendai virus envelope (see Fig. 1) is composed of a lipid bilayer with a matrix protein (M, MW 38 000) on the inside and two different transmembrane proteins, the hemagglutinin-neuraminidase (HN, MW 68 000) and the fusion protein (F, MW 65 000), forming spikes on the outside. The base sequences of the genes coding for

the three Sendai virus membrane proteins M, F, and HN have been determined2-4. Other structural components of the virus particle are the nucleoprotein (NP), the most important element of the nucleocapsid, the polymerase (P) and a group of undefined proteins, called large proteins (L). The HN protein is responsible for adsorption of virions to the infected cell. The HN protein of Sendai virus consists of tetramers and dimers which are connected by disulfide bridges. The size of the HN proteins varies for different paramyxoviruses. The F protein is smaller in size than the HN molecule. A biologically inactive precursor molecule, designated F,, is produced during infection. This precursor is processed to the biologically active fusion protein via proteolytic cleavage by host cell enzymes after an arginine residue, to two disulfidelinked subunits F, and F,. The biologically active F protein is involved in cell fusion, hemolysis and virus penetration in the host cell. There is no major variation in the size of the F protein in different paramyxoviruses. Detergents Detergent extraction is a commonly used first step in the purification of a membrane protein. Detergents (amphiphiles, surfactants) are lipid-like substances. They possess a hydrophilic head and a hydrophobic tail and are able to compete with the lipids in a bilayer. Addition of an excess of detergent to a virus preparation may result in extraction of the integral membrane proteins from the lipid bilayer. Subsequent ultracentrifugation provides a supernatant containing a mixture of membrane proteins and detergent molecules. There are (a) mild non-ionic detergents, e.g. the Triton series, octylglucoside; (b) mild amphoteric detergents, e.g. 3-[(3-cholamidopropyl)-dimethylaminol-l-propane sulphonate (CHAPS); (c) b’l 1 e salts, e.g. cholate, taurodeoxycholate, which are naturally occurring detergents that resemble CHAPS in having a similar steroid structure and generally have a more profound effect on the structure of the protein; and (d) denaturing, ionic detergents, e.g. sodium dodecyl sulphate (SDS). Several of these detergents were used in the studies described below. HPLC of Sendai virus membrane proteins Several modes of HPLC can be used for the purification of membrane proteins provided that the elution conditions are adjusted to the special requirements of these proteins, i.e. presence of a certain concentration of organic solvent, SDS, urea, guanidine-HCl or a non-ionic detergent. We have used the following modes of HPLC for the purification of

trends in analytical chemistry, vol. 5, no. 9,1986

A280 0.03

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Fig. 2. SE-HPLC of a detergent extract of Sendai virus. Purified virions (20 mg of virus protein) were extracted with different detergents at a final concentration 2% (w/w) for20 min at room temperature. The detergent-to-viral protein ratio was 2 (wlw). After centrifugation at 100 000 g, extracted viral proteins HN and F werepresent in the supernatant. The Zorbax GF-450 column (two 25 cm x 9.4 mm I.D. columns in tandem) was eluted with 50 mM sodium phosphate, pH 6.5, containing 0.1% SDS. The flow-rate was 1 mllmin and the absorbance was monitored at 280 nm. The F protein is indicated by an asterisk. V, = 14.2 ml and V, = 26.6 ml. A, Triton X-100; B, octylglucoside; C, decylpolyethyleneglycol; D, CHAPS; E, taurodeoxycholate.

Sendai virus proteins: (1) size-exclusion (SE) HPLC, (2) ion-exchange (IE) HPLC and (3) reversed-phase (RP) HPLC. Size-exclusion HPLC Several types of column packings are available for separation of membrane proteins according to size’. Since separation is only possible between the void volume and the total volume of the column, the peak capacity is rather low and SE-HPLC should therefore preferably be combined with other modes of HPLC. To prevent aggregation, a detergent has to be present during SE-HPLC of membrane proteins. SDS is at present used most frequently for this purpose. Non-ionic detergents are the most suitable additives when further purification by IE-HPLC is required.

227 Fig. 2 shows an example of a separation according to size of a Sendai virus membrane protein mixture using 0.1% SDS in the elution buffer. At the same time this figure shows the potential of five different detergents, which were used under identical conditions, to extract the membrane proteins from the virus envelope. We used (A) Triton X-100, (B) octylglucoside, (C) decylpolyethyleneglycol-300 (decylPEG), (D) CHAPS and (E) taurodeoxycholate. To facilitate a comparison, the F protein is indicated by an asterisk. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that HN was present in the peaks which were eluted at 15.4 and 16.2 min. These peaks represent the tetramer and the dimer of HN with MW 136 000 and 272 000, respectively. These results were found when Triton X-100, octylglucoside and decyl-PEG were used for extraction. In contrast to this, taurodeoxycholate and CHAPS also released the M protein and the peaks preceding F contained aggregates of HN, F and M. In all cases, more F protein is extracted than HN although it has been found by immunological methods’ that the ratio HN:F is 2 in the intact virion. Instead of 0.1% SDS, non-ionic detergents or an organic solvent can be included in the elution buffer5’6. This generally results in broader peaks, but it is particularly useful in multidimensional HPLC. SEHPLC may be followed by IE- or RP-HPLC. The non-ionic detergents octylglucoside, decyl-PEG and others which do not interfere with the detection of protein at 280 nm are suitable for inclusion in buffers when SE- and IE-HPLC are combined. Ion-exchange HPL C IE-HPLC of membrane proteins requires the inclusion of a non-ionic detergent in the elution buffer. Concentrations in the range 0.03-2.0% have been reported in the literature. We have used 0.1% Triton X-100 in a 20 mA4 Tris buffer, pH 7.8 and detection at 275 nm to avoid the high UV absorbance due to the phenyl ring in Triton X-1007. HN and F were eluted by a salt gradient. The HN protein eluted first in several peaks and the F protein eluted at a higher salt concentration as a broad peak consisting of multiple peaks (see Fig. 3). The multiple peaks observed for both proteins might be due to different aggregates or polymeric forms (tetramer and dimer of HN) but also to differences in charge caused by acidic carbohydrate moieties attached to HN and F. More than 75% of the oligosaccharides from F protein are acidic while 18% of the oligosaccharides from HN protein are acidic. Most of the gradient elution systems used in IEHPLC are not harmful to. the structural conformation of the protein and IE-HPLC, if necessary com-

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I

A275 0.01

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Fig. 3. Anion-exchange HPLC of a Triton X-100 extract of purified Sendai virions. The Mono Q column (Pharmacia, Sweden) was eluted with a 24-min gradient from 20 mM Tris-HCI, pH 7.8, containing 0.1% Triton X-100, to 0.5 M sodium chloride in the same buffer. The flow-rate was 1 mllmin and the absorbance was monitored at 275 nm. Fractions were analyzed by SDS-PAGE (10% gels). The molecular weight of reference proteins (ref) is given in kilodalton. extr = the Triton extract. (From ref. 7.)

bined with SE-HPLC, therefore is a suitable method for the purification of an intact, biologically active membrane protein. Reversed-phase HPLC Many proteins unfold upon contact with the hydrophobic ligands of a reversed-phase column support and/or by being dissolved in the elution buffer, which is generally an aqueous organic solvent of low pH. Therefore it is conceivable that all of the hydrophobic groups are available for interaction with the hydrophobic ligands. An average protein’ contains 20.2% large hydrophobic amino acids: leucine, valine, isoleucine, methionine (LVIM is the one-letter code for these amino acids). The Sendai virus proteins are considerably more hydrophobic, having 25.7-30.3% LVIM. The approximate number of sites which may interact with the column calculated from the percentage LVIM for a typical reference protein ribonuclease (14.5%) and the Sendai virus M protein (30.3%) is 18 and 103, respectively. When we also include tyrosine, tryptophan and phenylalanine, these numbers are 27 and 132. The exact number is even higher, since the alkyl part of lysine will also interact with a hydrophobic ligand. This example not only shows that a protein is a multi-point attachment solute but also that a membrane protein molecule does contain a considerably higher number of hydrophobic groups available for interaction with reversed-phase column particles than a hydrophilic protein. Elution is achieved with an increasing concentra-

tion of organic solvent and relatively high concentrations are needed to break up the many contacts of integral membrane proteins with the ligands. Proteins may be precipitated by an organic solvent and since membrane proteins require relatively high concentrations of organic solvent for elution, they are balanced between two opposing conditions: elution by an increasing concentration of organic solvent up to 50% and precipitation of the protein by organic solvent. These conditions play an important role in the outcome of an RP-HPLC purification of a membrane protein. A third important parameter is the size of the protein. Generally, as has been shown by Rubinstein’, the larger the protein the higher the concentration of organic solvent required for its elution. Therefore it is not unexpected that RP-HPLC purifications of small membrane proteins are more successful than those of larger proteins.

TABLE I. Hydrophobicity proteins.

(% LVIM) and molecular weight of

Protein

MW

% LVIM

Sendai virus F, F, M HN Bovine ribonuclease Ovalbumin

13 000-15 000 50 000 38 000 68 000 13 700 43 000

29.7 29.6 30.3 25.7 14.5 27.0 20.2

Average

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EX

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2345678910

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I

I

I

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Fig. 4. RP-HPLC of a Triton X-100 extract ofpurified Sendai virions which was reduced with dithiothreitol. The Phenyl-SPW RP (Toy0 Soda, Japan) column (50 x 4.6 mm) was eluted with a 24-min gradient, consisting of 15-75% acetonitrile in water containing 0.05% trifi’uoroacetic acid. The flow-rate was 1 mllmin and the absorbance was monitored at 214 nm. Fractions (I-10) were analyzed by SDSPAGE (13% gels). The molecular weight of reference proteins (REF) is indicated in kilodaltons. EX = the Triton extract. Dotted area, HNprotein; hatched area, Ft protein. _ _ _

Sendai virus membrane proteins are an excellent example of such a purification problem. To avoid precipitation, it was necessary to reduce the disulfide bridges in proteins in a detergent extract of Sendai virus. The tetramer and the dimer of HN were reduced to the monomeric form (MW 68 000) and F was reduced to F, (MW 50 000) and F, (MW 13 000-15 000). Reduction does not affect the size of the M protein (MW 38 000). The resulting mixture contains hydrophobic membrane proteins (MW 13 000-68 000). The hydrophobicities (percentage LVIM) and the molecular weights are given in Table I and compared with the corresponding data of two reference proteins bovine ribonuclease and ovalbumin and an average protein’. The mixture containing F,, F2, M and HN was subjected to RP-HPLC on a Phenyl-SPW RP column. Phenyl-5PW RP is a hydrophilic resin-based support with pores of 1000 A of which the surface is covered with a high density of phenyl groups. The elution pattern is shown in Fig. 4 together with an SDS-PAGE analysis of the indicated fractions 1 to 10. F, and M which are relatively small and have similar hydrophobicities (see Table I), are both eluted as a sharp peak at 32.5 and 40% organic solvent, respectively. This difference shows the importance of the size of the protein, MW 13 000-15 000 vs. MW 38 000. The two larger Sendai virus membrane proteins are eluted at higher organic solvent concentrations, between 44.5 and 52.5%, as multiple peaks (HN, dotted area; F,, hatched area). HN is eluted earlier than F, (44.5 vs.

48% organic solvent). The lower percentage of large hydrophobic amino acids (25.7 vs. 29.6%) appears to be more important than the larger size of HN. The multiple peaks may be caused by repeated precipitation and dissolution of the larger membrane proteins . Earlier studies with RP-HPLC columns with smaller pore sizes (100,250 and 300 A) and other ligands (C, and C,,) resulted in similar elution patterns although the yields of F, and HN were much lower, especially with the 100-A column7>*‘. These studies, in which a similar membrane protein sample was allowed to interact with different ligands, Ci, C,, and Phenyl, and from which the results showed that the proteins were eluted at almost identical organic solvent concentrations, justify the conclusion that the strength of the interaction is largely dependent on the solute and not on the hydrophobic nature of the ligand. The large number of interaction sites in a protein molecule already mentioned above is crucial for hydrophobic interaction and subsequent elution. So far it has not been possible to achieve elution with lower organic solvent concentrations by using columns with ligands which are less hydrophobic than C,,, e.g. C, and Phenyl. Yields were not significantly different when we studied reference proteins with these RP-HPLC columns. In contrast to this, yields of the large membrane proteins HN and F differed depending on the pore size of the column. They were excellent when the 1000-A column was used.

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In a recent study”, nine RP materials with various bonded phases were studied for the separation of hydrophilic proteins with two solvent systems. Proteins were eluted either with a gradient of acetonitrile in 0.05% trifluoroacetic acid or with a gradient of ethanol-n-butanol(4: 1, v/v) in 12 mM hydrochloric acid. Four equally well performing columns were selected for the purification of Sendai virus membrane proteins. In this case distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent system. Conclusion In conclusion, these studies with the hydrophobic membrane proteins of Sendai virus as a model have shown that SE-HPLC is generally of limited value but can be successfully used to purify small amounts of membrane proteins by collecting only part of a peak sufficiently far away from neighboring peaks. Preferably, SE-HPLC should be used in combination with other modes of HPLC. The extreme hydrophobicity of large integral membrane proteins (MW > cu. 40 000) is a serious drawback for the application of RP-HPLC in their purification. These proteins can be purified by RPHPLC but recoveries are often low and success may depend on the combination of column and solvent system. Other ligands which are far less hydrophobic have to be developed to reduce the dominant disproportionate contribution of the protein molecule to the hydrophobic interaction. IE-HPLC will be most useful for purification of all sizes of membrane proteins and the mild conditions

used for elution may provide an intact membrane protein. Moreover, a wide variety of detergents, salts and pH values can be employed to obtain the optimal elution conditions.

References 1 C. Orvell and E. Norrby, in M. H. V. van Regenmortel and A. R. Neurath (Editors), Immunochemistry of Viruses, Elsevier, Amsterdam, 1985, p. 241. 2 Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda and H. Shibuta, Nucleic Acids Res., 12 (1984) 7965. 3 B. M. Blumberg, C. Giorgi, K. Rose and D. Kolakofsky, J. Gen. Virol., 66 (1985) 317. 4 B. Blumberg, C. Giorgi, L. Roux, R. Raju, P. Dowling, A. Chollet and D. Kolakofsky, Cell, 41(1985) 269. 5 G. W. Welling, K. Slopsema and S. Welling-Wester, 1. Chromatogr., 359 (1986) 307. 6 G. W. Welling, G. Groen, K. Slopsema and S. WellingWester , J. Chromatogr., 326 (1985) 173. 7 G. W. Welling, J. R. J. Nijmeijer, R. van der Zee, G. Groen, J. B. Wilterdink and S. Welling-Wester, J. Chromatogr., 297 (1984) 101.

8 M. 0. Dayhoff, L. T. Hunt and S. Hurst-Calderone, in M. 0. Dayhoff (Editor), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, 1978, Vol. 5, suppl. 3, p. 363. 9 M. Rubinstein, Anal. Biochem., 98 (1979) 1. 10 R. van der Zee, S. Welling-Wester and G. W. Welling, J. Chromatogr., 266 (1983) 577. 11 R. van der Zee, T. Hoekzema, S. Welling-Wester and G. W. Welling, J. Chromatogr., (1986) in press. Gjalt W. Welling, Ruurd van der Zee and Sytske Welling-Wester are at the Medical Microbiology Department of the University of Groningen, Oostersingel 59, 9713 EZ Groningen, The Netherlands. Their research interests are in the isolation and characterization of proteins which are important in the field of medical microbiology.

trends

Nuclear magnetic resonance detection for the on-line identification of liquid chromatography eluents David A. Laude, Jr. and Charles L. Wilkins Riverside, CA, U.S.A.

Although the analytical capability of gas chromatography (GC) has been enhanced by coupling to information-rich detectors such as mass and infrared (IR) spectrometers, incompatible sample require-

ments have hindered the development systems for on-line identification of ance liquid chromatography (HPLC) recently, following intensive technical has HPLC-mass spectrometry (MS) mercially viable, while the realization uid chromatography-IR continues to

of analogous high-performeluents. Only development, become comof on-line liqbe plagued by