Hydroxylapatite chromatography of protein-sodium dodecyl sulfate complexes. A new method for the separation of polypeptide subunits.

Abstract Hydroxylapatite (calcium phosphate) chromatography was investigated as a method for separating protein subunits dissociated with sodium dodecyl sulfate (SDS). The proteins used included: a series of 11 well characterized proteins composed of single or identical subunits varying in molecular weight from 11,700 to 165,000; hemoglobin, composed of α and β chains of 146 and 141 amino acids; and vaccinia virus structural proteins, composed of more than 15 different polypeptide subunits. Reduced proteins were complexed with SDS and adsorbed to columns of hydroxylapatite. Using linear gradients of sodium phosphate, pH 6.4, containing 0.1% SDS and 1mm dithiothreitol, all tested proteins eluted between 0.2 and 0.5 m phosphate. A useful feature of the method is that proteins do not all elute in order of molecular weight. Thus polypeptide separations on hydroxylapatite are different from those obtained by polyacrylamide gel electrophoresis or gel filtration in SDS. The high resolution of the method was demonstrated by separating the α and β chains of hemoglobin and complex mixtures of viral polypeptides. By using polyacrylamide gel electrophoresis and hydroxylapatite chromatography in succession, it has been possible to separate vaccinia virus structural polypeptides that had not previously been resolved.


Hydroxylapatite
(calcium phosphate) chromatography was investigated as a method for separating protein subunits dissociated with sodium dodecyl sulfate (SDS). The proteins used included: a series of 11 well characterized proteins composed of single or identical subunits varying in molecular weight from 11,700 to 165,000; hemoglobin, composed of a and fi chains of 146 and 141 amino acids; and vaccinia virus structural proteins, composed of more than 15 different polypeptide subunits. Reduced proteins were complexed with SDS and adsorbed to columns of hydroxylapatite.
Using linear gradients of sodium phosphate, pH 6.4, containing 0.1% SDS and 1 mM dithiothreitol, all tested proteins eluted between 0.2 and 0.5 M phosphate.
A useful feature of the method is that proteins do not all elute in order of molecular weight.
Thus polypeptide separations on hydroxylapatite are different from those obtained by polyacrylamide gel electrophoresis or gel filtration in SDS. The high resolution of the method was demonstrated by separating the LY and fl chains of hemoglobin and complex mixtures of viral polypeptides.
By using polyacrylamide gel electrophoresis and hydroxylapatite chromatography in succession, it has been possible to separate vaccinia virus structural polypeptides that had not previously been resolved.
Polyacrylamide gel electrophoresis has been established as a useful method for separating protein subunits dissociated with sodium dodecyl sulfate (1). At concentrations of SDS' used for electrophoresis, different proteins bind identical amounts of SDS on a gram to gram basis (2, 3) and have hydrodynamic properties that are a unique function of polypeptide chain length (4). For these reasons, the electrophoretic mobilities of saturated SDS complexes are dependent on the molecular weight of the protein portion (5, 6).
We now report a new way of separating protein subunits in SDS. This consists of adsorbing proteill-SDS complexes onto 1 The abbreviations used are: SDS, sodium dodecyl sulfate; DTT, dithiothreitol. hydroxylapatite and eluting with 0.1% SDS, 1 rnht DTT, and a gradient of sodium phosphate.
Although the total concentration of SDS remains constant, the equilibrium monomer concentration of SDS drops as the ionic strength of the grad ent increases.
A useful feature of this system is that proteins do not all elute in order of molecular weight.
Thus, polypeptide separations on hydrosylapatite are different from those obtained by polyacrylamide gel electrophoresis or gel filtration in SDS.

EXPERIMENTAL PROCEDURE
Materials-Hydrosylapatite (Rio-Gel HT) was obtained from Bio-Rad Laboratories; sodium dodecyl sulfate was the special13 pure grade from Mann and dithiothreitol came from Calbiothem.
All other chemicals were reagent grade. Rabbit globin, labeled with ['*C]histidine, was a gift of Dr. W. French Anderson.
Preparation of Protein-SDS Complexes-Radioactively labeled proteins were dissolved in 0.01 M sodium phosphate, pH 6.4-l 9 SDS-l $T, mercaptoethanol and placed in a boiling water bath for 2 min. The samples were then diluted lo-fold with 0.01 M sodium phosphate, 1~1-1 6.4-O.lCJ, SDS and applied directly to hydroxylapatite columns. Unlabeled proteins, at a concentration of 4 mg per ml, were complexed with SDS in a similar manner except that 2yc SDS and 2% rnercaptoethanol were used and the samples were dialyzed overnight instead of being diluted.
C%roma!ography-Iydroxylapatite was washed with 0.01 M sodium phosphate, pH 6.4-0.1cA~ SDS-l mM DTT. Columns (0.9 x 20 cm) were poured over a 0.5.cm layer of fine grade Sephadex G-25. Protein samples, from 1 to 30 ml in volume, were applied and washed in with 1 to 2 column volumes of the 0.01 M phosphate-SDS-DTT solution.
Linear gradients were formed with a simple two-chambered device and flow rates of 5 to 15 ml l)er hour were obtained by gravit.y. Sixty drop frac- The gradient marker contained 100 ml of 0.2 M sodium phosphate, pH 6.4-0.170 SDS-1 rnM DTT in one chamber and 100 ml of 0.5 M sodium phosphate, pH 6.4-0.170 SDS-l mM I>TT in the other. tions, approxima.tely 1.5 ml, were collected and portions were either counted in a toluene based scintillation fluid containing appropriate amounts of Triton X-100 and HZ0 to form a stable emulsion or the ABso was determined.
Hydroxylapatite columns were only used once. Refractive indexes, measured on every tenth fraction, were converted to phosphate molarity with a standard curve. Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electrophoresis in O.lyO SDS was performed as previously described (7) using the procedure of Maize1 (1). After electrophoresis the gels were frozen and sliced into l-mm sections and then dissolved with hydrogen peroxide and counted (8) or eluted overnight with 1 ml of 0.1 y0 SDS-O.01 M sodium phosphate, pH 6.4-l InM DTT. When polyacrylamide gel electrophoresis was to be followed by hydroxylapatite chromatography, the pooled fractions were made 1 'z in mercaptoethanol, heated at 100" for 2 min, allowed to cool to room temperature, and applied directly to hydroxylapatite columns. were resolved with a linear phosphate gradient (Fig. 1). Upon re-chromatography, the separated peaks eluted at 0.354 M and 0.380 M phosphate, respectively (Fig. 2). Larger (10 mg 3. Hydroxylapatite chromatography of 14C-amino acidlabeled vaccinia virus structural polypeptides.

Separation of o( and p Hemoglobin
The gradient was made as in Fig. 1. The IZoman numerals are used to identify t,he pooled fractions.
hydroxylapatite to obtain sufficient material to identify the o( and fi chains, which have different leucine to iaoleucine ratios. Amino acid analysis indicated that the a! chain eluted first and the fl chain second. Human hemoglobin separated in an ahnost identical manner. Separation of Vaccinia Virus Structural Polypeptides-Vaccinia virus contains a large number of structural polypeptides which are incompletely resolved by polyacrylamide gel electrophoresis in SDS (9, 10). 14C-Amino acid-labeled vaccinia virus structural polypeptides were completely adsorbed to hydroxylapatite.
Upon elution with a 0.2 to 0.5 M phosphate gradient containing SDS and DTT, eight peaks were resolved (Fig. 3). Preliminary experiments with a 4-cm column gave poorer resolution. The pooled fractions, indicated by Roman numerals I to V1 (Fig. 3) Fig. 3 were dialyzed overnight against water, lyophilized, dissolved in SDS and mercaptoethanol, mixed with total aH-amino acid-labeled vaccinia virus structural polypeptides, heated at 100" for 2 min, and subjected to electrophoresis on 7.5% polyacrylamide gels. The gels were sliced and counted as described.
The Roman numerals refer to the fractions pooled in Fig. 3.

chromatography
(labeled with 14C-amino acids) also were mixed with unfractionated vaccin a virus structural polypeptides (labeled with 3H-amino acids) and then analyzed by SDS polyacrylamide gel electrophoresis. It was apparent, from the double-label experiments, that each hydroxylapatite peak contained a unique group of viral polypeptides (Fig. 4). Furthermore, three polypeptides with very similar electrophoretic mobilities were found in hydroxylapatite Peaks II, V, and VI. The separation of polypeptides with similar electrophoretic mobilities by hydroxylapatite chromatography was directly demonstrated in the following experiments. Vaccinia virus polypeptides, labeled with [311]tryptophan and r4C-amino acids were separated by polyacrylamide gel electrophoresis into three molecular weight groups designated by A, B, and C (Fig. 5). Upon chromatography, the polypeptides with lowest electrophoretic mobility (A) eluted as Peak VI; the polypeptides with ntermediate mobilities (B) eluted as Peaks II, V, and VI; and the most rapidly migrating polypeptides (C) eluted as Peaks I, II, III, and IV (Fig. 6). Differences in 3H:14C ratios suggest that the separated polypeptides have different amino acid compositions. A appears to be composed of unresolved polypeptides with different contents of tryptophan.
Although viral polypeptides separated quite differently by hydroxylapatite chromatography and polyacrylamide gel electrophoresis, polypeptides with lower electrophoretic mobilities tended to elute at higher molarities of phosphate.
Chromatography identical subunits, was exa.mined by hydroxylapatite chromatography in SDS (Fig. 7). Most eluted as a single major peak, although bovine serum albumin and ovalbumin appeared somewhat heterogeneous.
All  and 0.5 M phosphate and no correlation with molecular weight was found.
For example, polypeptides ranging in molecular weight from 11,700 (cytochrome c) to 165,000 (thyroglobulin) eluted at lower molarities of phosphate than serum albumin which has a molecular weight of 68,000.
Lysozyme, serum albumin, and ovalbumin, which are composed of single polypeptide chains, were also chromatographed without SDS. All eluted at less than 0.15 M phosphate, indioating that these proteins bind less strongly to hydroxylapat te in the absence of SDS.

DISCUSSION
These studies demonstrate the usefulness of hydroxylapatite chromatography in SDS as a method for analytical and preparative separations of protein subunits.
Since polypeptides are not all eluted from hydroxylapatite in order of molecular weight, the separations are different from those obtained in SDS by polyacrylamide gel electrophoresis or gel filtration. Bernardi and Kawasaki (13) suggested that carbosyl groups (or in special cases phosphate groups), which are locaated at the surface of native proteins, are responsible for binding to hydrosylapatite.
They also found that proteins denatured with urea are less well retained or not retained at all by hydrosylapatite columns equilibrated with 1 mM phosphate buffers.
This was attributed to a decrease in the number of carbosyl groups on the surface of proteins in the random coil configulution (13).
In contrast we find that proteins denatured with SDS bind more strongly than native proteins to hydrosylapatitr.
The difference in adsorption of proteins in urea and SDS to hydroxylapatite may be explained by the very different states of the proteins in the two types of solvents.
At sufficiently high monomer concentrations of SDS (greater than 5 X 10h4 ar) a wide variety of proteins bind identical amounts of SDS (3). A comples with a stoichiometry of 0.4 g of SDS per g of protein is formed between 5 and 8 X 1OP M SDS monomer (3). A second complex, which is saturated at 1.4 g of SDS per g of proteins is observed above 8 X 1OP M SDS monomer (3). The saturated protein-SDS complexes exist as rod-like molecules, the length of which varies uniquely with the molecular weight of the protein moiety (4). Presumably, the large amount of SDS bound or the altered conformation of the protein is responsible for the strong attachment to hydroxylapatite. In aqueous solutions SDS exists as monomer and micellar aggregates, but only the monomer form binds to proteins (3). We considered that the amount of SDS bound to proteins would decrease at phosphate (sollcelltrations above 0.2 M since the equilibrium monomer concentration of SDS is dependent on ionic strength (3, 14). All proteins tested eluted from hydroxylapatite at phosphate concentrations between 0.2 and 0.5 RI. Although it, is clear that proteins ;\re not all eluted in order of molecular weight, the factors responsible for protein separations are not understood. It is possible that at low SDS monomer concentrations, differential binding of SDS or differences in the conformations of proteins become significant.