An improved method for the preparation of renaturable apohemoglobin chains

ANALYTICAL

BIOCHEMISTRY

117, 103-107 (1981)

An Improved Method for the Preparation Renaturable Apohemoglobin Chains FRED K. FRIEDMAN, Laboratory

of

KENNETH

ALSTON,

AND ALAN

of

N. SCHECHTER

Chemical Biology, National Institute of Arthritis. Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received June 2, 1981

A rapid, effective procedure for the preparation of renaturable globin chains from human hemoglobin is described. The method utilizes hydrophobic interaction chromatography in an acid medium to separate the a and fi chains of apohemoglobin. Rapid procedures for reconstitution of hemoglobin from these chains and hemin are also presented.

Reconstitution of hemoglobin from the apoprotein and hemin, or from its constituent globin chains and hemin is performed both as a model system to study the process of protein folding and assembly, and as a method for the construction of hemoglobins selectively modified in either the globin or heme. Many such studies have been carried out (reviewed in Refs. (l-3)) and have provided insight into the structural basis of numerous aspects of hemoglobin function. The experiments require conversion of hemoglobin into globin( manipulation of globin and porphyrin, and recombination of all components to yield hemoglobin. Procedures are available for the preparation of apohemoglobin (4,5), isolated globin chains (6), and assembly intermediates (7,8). In contrast to the holoproteins, the corresponding apoproteins are relatively unstable and must be used shortly after their isolation. Furthermore, the preparation of renaturable, separated globin chains is a lengthy procedure that involves the reaction of hemoglobin sulfhydryls with p-mercuribenzoate to induce subunit dissociation, chromatographic separation of subunits, regeneration of sulfhydryl groups, and heme removal (8). We report a new method in which heme

is initially removed from hemoglobin to yield apohemoglobin, cy”$.’ The constituent (Y’ and $ chains are dissociated in acid and separated by hydrophobic interaction chromatography (9- 11). This application extends the range of this separation technique to preparation of the constituent chains of a multisubunit protein. Furthermore, the separation is rapid and, together with rapid reconstitution procedures, yields hemoglobin which has properties identical to the native form, as tested by several criteria. MATERIALS

AND METHODS

Preparation of hemoglobin. Hemoglobin A was obtained from lysis of human erythrocytes and was purified by ion-exchange chromatography essentially according to Huisman and Dozy (12), using DEAE-Sephacel (Pharmacia). Conversion to methemoglobin was accomplished by reaction with 20% excess of potassium ferricyanide for 30 min and filtration through a Sephadex G-25 (Pharmacia) or Bio-Gel P-2 (Bio-Rad) column equilibrated with water. Preparation of apohemoglobin. Heme was removed from methemoglobin by the method ’ A zero superscript following a or @ indicates that the subunit is lacking heme. 103

0003-2697/81/150103-OSSO2.00/0

104

FRIEDMAN,ALSTON,ANDSCHECHTER

of Teale (5). An ice-cold 1% solution of methemoglobin was titrated to pH 2.0 with cold 1 M HCI, and heme was extracted by mixture with an equal volume of cold methylethylketone (Baker) at 0°C in a separatory funnel. The extraction procedure was repeated to remove residual hemin from the aqueous layer. Maintenance of temperature at 0°C is essential for rapid phase separation, and is achieved by cooling the methylethylketone to subzero temperatures prior to its additon to protein. The protein solution was freed of dissolved ketone and acid by filtration through a Sephadex G-25 column equilibrated with 0.01 M NaH2P04, 0.2 mM EDTA. The apohemoglobin thus obtained is stable at 4°C for several days. Separation of globin chains. This procedure was performed at room temperature. The pH of the desired amount of apohemoglobin was lowered to pH 3.0 by dropwise addition, with stirring, of 1 M phosphoric acid. One-fourth volume of 2.0 M NaCl was then added. The resulting solution was applied to a 3.0-cm-diameter octyl agarose (Miles-Yeda) column equilibrated with 0.40 M NaCl, 0.01 M sodium phosphate, pH 3.0. The amount of protein and the column height was such that the loading ratio was 2 mg protein/ml bed volume; 200-300 mg protein was usually loaded. Under these conditions the protein is adsorbed onto the gel. After washing with 2 bed vol of the equilibration buffer, (Y’ was eluted by applying 3 bed vol of 0.07 M NaCl, 0.01 M sodium phosphate, pH 3.0. Application of 0.01 M sodium phosphate, pH 3.0, then eluted p”. The resin was then washed with water and stored in the cold between uses, Reconstitution of hemoglobin. Two routes were followed for reconstitution, differing according to whether hemin was added to the chains after or prior to their recombination. Protein concentrations were determined spectrophotometrically using the molar extinction coefficients at the uv spectra maxima (6); these are 1.OO, 1.54, and 1.31

X lo4 M-’

cm-’ for cy”, p”, and cu”$, respectively. A hemin (BDH) solution was freshly prepared by dissolving 1 mg/ml 0.01 M NaOH and removal of undissolved material by centrifugation; the concentration (approximately 1.5 mM) was calculated from the absorbance at 385 nm, using an extinction coefficient of 5.0 X lo4 M-’ cm-’ (13). All buffers during reconstitution contained 0.2 mM EDTA. All operations were performed at 4°C unless noted otherwise. The first reconstitution route entails reassociation of chains to form apohemoglobin, followed by hemin addition. The chains were mixed in an equimolar ratio and dialyzed overnight against 0.01 M potassium phosphate, pH 7.0. Any precipitated material was removed by centrifugation. Hemin (1.25 equivalents) was then added at 0°C. After 2 h the solution was dialyzed against 0.01 M Tris-HCl, pH 8.5. The material was then absorbed on a DEAE-Sephacel column (2.5 X 2 cm for 100 mg protein) equilibrated with the dialysis buffer and washed with 4 bed vol of the same. The reconstituted hemoglobin was then eluted with 0.02 M potassium phosphate, pH 7.5, and dialyzed against 0.1 M potassium phosphate, pH 7.0. The second reconstitution route involves hemin addition to each separate chain, followed by recombination of the individually reconstituted subunits. Because the isolated subunits in the unliganded ferric state are unstable (7,14,15), the more stable cyano derivatives were produced. Each globin chain, in ice, was titrated to pH 5.5-6.0 with 1 M NaOH. Hemin (1.25 equivalents) mixed with a 50-fold excess of solid KCN was added and the pH adjusted to pH 6.0 with 1 M HCI. After 2 h at 0°C the reaction mixtures were dialyzed overnight versus 0.1 M potassium phosphate, pH 7.0. The o-chain mixture contained precipitate and was clarified by centrifugation. The reconstituted (Y and 6 chains were then mixed in equimolar ratio, using a molar absorbancy for each chain of 1.1 X lo4 cm-’ at 540 nm. The

PREPARATION

OF APOHEMOGLOBIN

105

CHAINS

mixture was then dialyzed against 0.01 M Tris-HCl, pH 8.5, and purified with DEAESephacel as in the first reconstitution route. Characterization of proteins. Absorption spectra were taken on a Cary 17D spectrophotometer. Globin chains were identified by their electrophoretic mobility under denaturing conditions. About 5 pg of protein which had been dialyzed versus 6 M urea, 0.01 M-Tris acetic acid, pH 4.8, was applied to a well of a 5% polyacrylamide gel slab composed of a 1:30 ratio of bisacrylamide to acrylamide, 0.43% N,N,N’.N’-tetramethylethylenediamine (TEMED),’ and 0.075% ammonium persulfate (all electrophoresis reagents from Bio-Rad). The gel and running solutions were 6 M urea, 0.1 M acetic acid adjusted to pH 4.8 with Tris (Schwarz/ Mann). Electrophoresis proceeded overnight at room temperature at 60 V. Gels were stained for 1 h with 0.2% Coomassie brilliant blue R-250 in 50% methanol, 10% acetic acid; destaining was by diffusion into 10% methanol, 10% acetic acid. Electrophoresis under nondenaturing conditions was similar except for the use of 0.12% TEMED in the gel and 0.1 M Trisborate, pH 9.4, as the buffer. Protein samples which were made 5% in sucrose were electrophoresed at 4°C under 13 V/cm until the bromphenol blue marker dye had migrated to the end of the gel. This system resolves the separated subunits, hemoglobin, and their corresponding globins. Oxygen binding measurements were carried out with a Hem-O-Scan (Aminco). Deoxygenation curves were obtained by slowly passing nitrogen over the sample. Percent oxygenation at each oxygen pressure was determined from the difference in sample transmittances at 439 and 448 nm. Proteins were first concentrated in an Amicon ultrafiltration cell with a YM-10 membrane

and data were obtained for samples at a concentration of -0.5 mM at 20°C. A methemoglobin reductase system (16) was added to reduce methemoglobin and to maintain the protein in the ferrous state during the run. Cyanomethemoglobin was first reduced with a 20-fold excess of sodium dithionite under nitrogen for 5 min and then passed through a small column of Sephadex G-25 equilibrated with 0.1 M potassium phosphate, 0.2 mM EDTA, pH 7.0.

* Abbreviation ylethylenediamine.

phosphate was 250

used:

TEMED,

N.N.N’.N’-tetrameth-

RESULTS AND DISCUSSION

The fractionation of apohemoglobin into its constituent chains by octyl agarose is depitted in Fig. 1. Purity of the chains, assessed by

electrophoresis

in

acid-urea

gels

as

in

Fig. 2, demonstrates the efficiency of separation by this technique. I

3

22 E

1

:

AI-G1 10

FRACTION NUMBER

FIG. I. Separation of LY” and $ on octyl agarose. a’@’ (2.50 mg) was adsorbed onto a 3.0 X IS-cm column equilibrated with 0.4 M NaCI. 0.01 M sodium phosphate (pH 3.0). Collection of 25-ml fractions commenced and cue eluted upon washing with 0.07 M NaCI, 0.01 M sodium phosphate (pH 3.0). Application of 0.01 M sodium (pH ml/hr.

3.0)

at the

arrow

eluted

$.

Flow

rate

106

FRIEDMAN,

FIG. 2. Urea-acid gel of globins. the samples are OI’$, (Y’, $. Anode

ALSTON,

From left to right, is at the top.

The procedure is performed at pH 3.0, where a”/?” is predominantly dissociated into monomers ( 17). These are then separated by hydrophobic interaction chromatography (91 1), utilizing the differential affinity of each type of chain for the hydrophobic absorbent. High salt concentrations promote such interactions and induce adsorption of both chains to the gel in the initial 0.4 M NaCl solution. Subsequent lowering of the salt concentration by steps sequentially releases (Y’ and p”. The elution conditions should be determined empirically on small columns and samples. If too high a salt concentration is used in the first step, a0 slowly elutes as a small, broad peak; extensive washing of the column is then required to completely remove cy” and avoid its contaminating the $ fraction. If the first step contains too little salt, (Y’ elutes sharply but its trailing fractions include some $. Salt (0.07 M) was found to elute a sufficiently concentrated solution of cy” for subsequent work, and a wash with 3 bed vol was sufficient to completely desorb (x0. Each chain is obtained in 70 to 80% yield in this manner. These conditions have proven effective for the octyl agarose manufactured by Miles-Yeda, in which 15-20 rmol octylamine is coupled per milliliter of resin. Since the degree of substitution and type of gel matrix are expected

AND

SCHECHTER

to influence the capacity and adsorption characteristics of the gel, use of a different product may require a change in salt concentrations and/or elution volumes to optimize running conditions. This procedure has been used preparatively on 200 to 300 mg apohemoglobin. It can also be employed on an analytical scale with a few milligrams of protein and a small column; the heme extraction procedure is then most conveniently performed in a test tube in ice, and mixing of the phases is accomplished with a Vortex instrument for about 30 s. This separation method has been routinely applied to hemoglobin A, but has also been successful with hemoglobin S. It may also prove effective, in modified form, with other abnormal or animal hemoglobins, or perhaps with unrelated multisubunit proteins. The reconstitution procedure described is also rapid and effective. Since the isolated globin chains exhibit solubility minima at neutral pH (18), in the second route hemin is added to each chain at a pH which is sufficiently acid to ensure its solubility, yet near enough to neutrality for stability of the reconstituted hemoprotein. After a short reaction period, the proteins could be successfully transferred to neutral pH. Recombination of the reconstituted a and fl chains generated a nativelike hemoglobin, as judged by several criteria. The same is true for reconstitution through the first route, in which the globin chains are recombined in acid and dialyzed to neutral pH prior to hemin addition. Reconstituted hemoglobin resembled the native protein by several criteria: migration in polyacrylamide gel electrophoresis in 0.1 M Tris-borate, pH 9.4, and absorption spectra of the cyanomet forms in the uv, Soret, and visible regions. The half-saturation value for oxygen binding to native and reconstituted hemoglobin was 1 1.4 and 11.O Torr, respectively; the corresponding Hill coefficients were 2.65 and 2.46.

PREPARATION

OF APOHEMOGLOBIN

The ease with which hemoglobin may be broken down and reconstituted should make more accessible certain types of studies which in the past have contributed to our knowledge of structure-function relationships. Several reconstitution approaches have previously been described, the most efficient of which appears to be through half-filled intermediates in which a heme-containing subunit associates with its heme-deficient complementary subunit in an alloplex interaction (7,8). The specific problem at hand, however, may influence which approach is taken. This is exemplified in the instance that a reconstituted hemoglobin specifically modified in only one type of subunit is desired. If the heme moiety is to be altered in either the identity of the central metal or the porphyrin, the subunits are best reconstituted separately as in the second procedure. If a modification of the globin is desired, or if one type of chain in the final product is to be substituted by another type from the same or another species, the different chains are recombined with one another prior to coupling with hemin as in the first route. The half-filled intermediate route may also be taken in either situation. ACKNOWLEDGMENTS

CHAINS

REFERENCES 1. Asakura, T. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 52, pp. 447-455, Academic Press, New York. 2. Sano, S. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. 7, pp. 337-402, Academic Press, New York. 3. Hoffman, B. M. (1979) in The Porphyrins (Dolphin, D., ed.). Vol. 7, pp. 403-444, Academic Press, New York. 4. Rossi-Fanelli, A., Antonini, E., and Caputo, A. (1958) Biochim. Biophys. Actu. 30, 608-615. 5. Teale, F. W. J. ( 1959) Biochim. Biophys. Acto 35, 543. 6.

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We thank Dr. Y. K. Yip for helpful discussions, Miss Juanita Chambers for technical assistance, and Miss Laura Smith for assistance in preparation of the manuscript.

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