Reverse-Phase High-Performance Liquid Chromatography of Hydrophobic Proteins and Fragments Thereof’
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ANALYTICAL BIOCHEMISTRY 131,99-107 (1983) Reverse-Phase High-Performance Liquid Chromatography of Hydrophobic Proteins and Fragments Thereof’ GEORGE E.TARR*'~ ANDJOHN W.CRABB-~~ *Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, and lDepartment of Ophthalmology, University of Washington, Seattle, Washington 98195 Received January 31, 1983 Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LMI were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LMI was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02-o. 1 M hexafluoroacetone/ NH3, pH 7.2 for highly acidic peptides; and (2) 6 M formic acid/O.13 M trimethylamine, pH 1.5, vs 4 M formic acid/O.09 M trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterilication of serine and threonine residues and appear to be reversible by aminoethanol treatment. High-performance liquid chromatography, tides (3-7) and hydrophobic, membrane-as- particularly reverse-phase chromatography, sociated polypeptides (8- 14), methods for such has supplanted the more classical methods of fractionations are not yet well established. This peptide fractionation in many laboratories and report incorporates the results of studies on constitutes one of the most important ad- types of reverse-phase supports and various vances in the field of protein sequence anal- solvent effects into methods which have en- ysis (1). The use of reverse-phase HPLC for hanced the HPLC resolution and recovery of the purification of small peptides is well es- the integral membrane proteins cytochrome tablished ( l-2). However, larger peptides, P-450 and bovine rhodopsin and of large pep- especially those derived from membrane-as- tides derived from P-450 LM2. sociated proteins, often chromatograph poorly because of insolubility, aggregation, or irre- MATERIALS AND METHODS versible adsorption to reverse-phase supports. Beckman Model 332 gradient HPLC sys- Despite increasing reports of successful re- tems were employed for all chromatography. verse-phase HPLC purification of large pep- Peptides and proteins in the column effluent were detected with either a Hitachi 100-10 ’ Portions of this report were presented at the Inter- spectrophotometer with an Altex flow cell or national Symposium on HPLC of Proteins and Peptides, a Beckman 160 detector equipped with a Cd held in Washington, D. C., November 16-17, 1981. lamp and 229-nm filter. Peak areas were * To whom all correspondence should be addressed. 3 Present address: Institut fur Physiologische Chemie, quantified with a Hewlitt-Packard Model Lehrstuhl I Ruhr-Universitat, Universit&strasse 150, 4630 3380A integrator or by cutting and weighing. Bochum, West Germany. The following reverse-phase HPLC columns 99 0003-2697/83 $3.00 Copyright C, 1983 by Academic Press. Inc. All nghtr of reproductmn tn any form rescrvcd. 100 TARR AND CRABB were used: from Waters Associates, PBon- provided by Dr. M. J. Coon (University of dapak CN, PBondapak C18, PBondapak Michigan, Ann Arbor). The cytochrome P- Phenyl (30 cm X 3.9 mm with 1O-pm pack- 450 LM2 and LM4 had been prepared as pre- ing); from Beckman, 5-pm Ultrasphere ODS viously described (15) and contained small (25 cm X 4.6 mm); from The Separations amounts of the nonionic detergent Renex. Group, 5-pm Vydac Cl8 and lo-pm Vydac Delipidated bovine rhodopsin preparations C,s (25 cm X 4.6 mm and 5 cm X 4.6 mm); were kindly provided by Dr. J. C. Saari (Uni- from Alltech, IO-pm Hamilton PRP- 1 ( 15 cm versity of Washington, Seattle). Outer seg- X 4.1 mm). All columns contain a silica-based ment membranes of bovine retinal rods (ROS) packing except the Hamilton PRP-1 which were isolated essentially as described by Pa- consists of a poly(styrene/divinylbenzene) per-master and Dreyer ( 16). Purified ROS were matrix and all columns were packed by the delipidated by affinity chromatography on manufacturer. Solvents were HPLC grade Con A-Sepharose according to De Grip et al. from Burdick and Jackson. Aminoethanol, (17) using Chaps as the detergent. Standard formic acid, and cyanogen bromide were pur- methods were used for cyanogen bromide chased from Aldrich and sequenal grade tri- cleavage of cytochrome P-450 and Mb (18) fluoroacetic acid (TFA)4 from Pierce. Water and for performic acid oxidation of insulin was twice distilled. All chromatography was and RNase (16). done at room temperature. Relative recoveries were calculated from The hexafluoracetone (HFA) sesquihydrate peak areas recorded and integrated at 2 14 or used in these experiments came from three 229 nm, with adjustment for flow rate. Ab- sources-Aldrich, DuPont, and PCR Re- solute recoveries were determined by amino search Chemicals. Unfortunately, because of acid analysis of individual test solutions and its toxicity, none of these American compa- peaks collected. N-terminal amino acid se- nies presently offer this unique chemical al- quence analyses (20) and PTH-amino acid though PCR has indicated that they may re- identifications (21) were done according to sume production. Sigma markets a HFA deu- Tarr. terate which appears to be an adequate substitute. RESULTS Commercially available proteins used as test HPLC of Large Hydrophobic Polypeptides materials included bovine ribonuclease and Ternary Solvent Efects (RNase, Worthington), bovine hemoglobin (Hb, Sigma), sperm whale myoglobin (Mb, Chromatography of the integral membrane Sigma), and bovine insulin (Sigma). Other test protein cytochrome P-450 LM2 is shown on peptides included a basic 15-residue cyanogen several reverse-phase supports in Fig. 1. Of bromide peptide from cytochrome c desig- particular interest is the resolution into a mi- nated P 15, the dipeptide WE, the tetrapeptide nor and major, more hydrophobic, compo- PKGK, the hexapeptide LWMRFA, and the nent (Fig. 1). As these two forms have the cyanogen bromide peptides of Mb. Two ho- same molecular weight by SDS-polyacryl- mogeneous forms of rabbit liver microsomal amide gel electrophoresis, the same amino acid cytochrome P-450 (LM2 and LM4) were kindly compositions and the same N-terminal se- quence, one is presumably a secondary mod- 4 Abbreviations used: TFA, trifluoracetic acid; PTH. ification of the other. The chromatographic phenylthiohydantoin; Mb, myoglobin; Hb, hemoglobin; patterns of P-450 and solvent effects vary be- CB I, II, III, cyanogen bromide peptides of Mb; SDS, tween columns. With acetonitrile as eluting sodium dodecyl sulfate; HFA, hexafluoroacetone; MeCN, solvent all except the PBondapak CN separate acetonitrile; PIOH, propanol; ROS, rod outer segment membrane; Con A, concanavalin A; ODS. octadecylsi- at least partially the major and minor forms lane. of P-450 (Fig. 2). Retention of the membrane HYDROPHOBIC PEPTIDES 101 resolution over binary solvent systems (22). The effect of ternary solvent systems in our separation of a standard test mixture of pep- tides and proteins composed of RNase, Mb, B-chain insulin, and cyanogen bromide pep- tides of Mb was dramatic but unexpected (re- sults to be published elsewhere). Nearly all test materials eluted later when 2-propanol was added to the acetonitrile, this despite the well-documented greater eluting power of MeCN.2-PrOHI-ProH 2Ad I propanol relative to acetonitrile (7). In fact, (rBondapak Cl8 retention time appears to go through a max- < imum as the proportion of propanol in the acetonitrile is gradually increased, until at MeCN A f&- roughly 1: 1 (v/v) the retention time has re- turned again to that seen with acetonitrile MeCN.I-PrOH 5i/l _/ii alone. Increasing amounts of propanol in- variably broadened the peaks, though at lower Vydoc Cl8 I proportions (2-5: I), this was more than com- pensated by the spreading out of the effective separation range. On the PBondapak columns (120 A, average pore diameter) cytochrome IIIIL,ILIII P-450 LM2 was affected by propanol/aceto- 20 30 40 nitrile mixtures like the smaller and more hy- MINUTES drophilic standards; however, on the Vydac FIG. I. HPLC of holocytochrome P-450 LM2 on se- column (300 A, pore), while retention was lected reverse-phase supports. About 1 nmol of freshly again increased, the peaks were dramatically acidified protein was injected in 40 ~1 at 0% organic. All sharpened (Fig. I). This may reflect improved eluting solvents contained 0.1% TFA; the organic com- ponents are indicated. Flow rates were 0.7 ml/min (PBon- solubility, as this wider pore support required dapak) and 1 ml/min (Vydac) in order to approximate a higher concentration of organic solvent for constant linear velocity; following a programmed jump elution. to 40% organic at 2 min, a linear gradient of 1% ml/min Recoveries of P-450 LM2 from PBondapak was used in each case. Absorbance was monitored at 229 and Vydac columns were similar, with about nm, 1.0 AUFS. 2/3 of the injected protein eluting in the two peaks described above; we have evidence that proteins is shortest on the PBondapak CN col- more protein elutes broadly after the main umn and longest on the large pore Vydac C 18; peak although we cannot rule out that some the separation of heme and protein is least on is stuck irreversibly. The LM4 isozyme is much the PBondapak Phenyl column and greatest more readily precipitated by organic solvents on the Vydac C 18.