ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 145, 236-239 (1971)

The Removal of Organic Phosphates from Hemoglobin’

MICHAEL BERMAN, REINHOLD BENESCH; AND RUTH E. BENESCH

Department of Biochemistry, Columbia University College of Physicians & Surgeons, New York, New York lOOW

Received March 24, 1971; accepted April 8, 1971

It has been shown that pH is an important factor in the separation of 2,3-diphos- phoglycerate (DPG) from hemoglobin. Hemolyeates of pH 7.5, but not below pH 7, can be freed of phosphate by gel filtration in 1% hr. It was also demonstrated that the mobility of DPG itself on Sephadex G-25 varies greatly with pH. The abnormally high mobility in neutral solution where the molecule is highly charged can be ex- plained by electrostatic repulsion within the molecule, giving it an extended confor- mation.

The recognition that organic phosphate es- ters, notably 2,3-diphosphoglycerate (DPG) profoundly alter the oxygen affinity of hemo- globin (l-3) has led to many investigations for which the preparation of phosphate-free hemoglobin is essential. We have described two procedures for this purpose; i.e., gel fil- tration (3) and dialysis through two-dimen- sionally stretched Visking membranes (4). The former method has been reported to give inconsist,ent results in the hands of some workers (5) yielding hemoglobin prep- arations containing variable amounts of DPG.

Since we had previously found that DPG is bound to oxyhemoglobin at pH values below neutrality (4), it was suspected that the pH of t.he hemoglobin solution subjected to gel filtrat.ion would influence the extent of removal of the phosphate ester from the protein. This was tested by adjusting equimolar mixtures of phosphate-free hemo- globin and diphosphoglyceric acid, pre-

1 This work was supported by U. S. Public Health Service Grants HE-05791 and HE-96787 from the National Heart Institute and ES-69535 from the National Institute of Environmental Health Sciences and by grants from the American Heart Association and from Smith Kline and French Laboratories.

2 Recipient of a U. S. Public Health Research Career Award from the National Heart Institute.

pared as described previously (4), to pH 6.5, 7.0, and 7.5, respectively. After high- speed centrifugation, aliquots of these solu- tions were chromatographed on a 1.5 X 22-cm column of Sephadex G-25 Fine (vr, = 18 ml; Vt = 39 ml) which had been equilibrated with 0.1 M NaCl. The column was eluted with the same solvent at 15 ml/hr at +3”. The results are shown in Fig. 1. It is clear that at pH 7.5 DPG is completely separated from hemoglobin. At pH 7 some overlap is evident, while at pH 6.5 a major portion of the DPG travels with the mobility of hemoglobin. This represents hemoglobin-bound DPG, whereas the free DPG appears as a more slowly moving shoulder. The tighter binding at lower pH values must be due to the increas- ingly positive charge on the hemoglobin molecule since the negative charge of the phosphate ester remains essentially constant over the range investigated (4, 7).

These experiments were, therefore, ex- tended to human hemolyzates, which were adjusted to pH 6.5 and 7.5, respectively, clarified by centrifugation, and chromato- graphed on the Sephadex column described above. The phosphate content of the eluted hemoglobin is shown in Table I. The values are expressed as total phosphate. Thus, the maximum DPG content is one half of that shown. It is clear that all but negh-

236

REMOVAL OF ORGANIC PHOSPHATES FROM HEMOGLOBIN 237

Effluent Volume in ml.

FIG. 1. Gel filtration of mixtures of Hb and DPG. Mixtures of 2 hmoles of hemoglobin and 2 rmoles of DPG in a volume of 3 ml were applied to the column. The solid lines represent hemoglobin concentration measured by the optical density at 540 rnp after con-

version to methemoglobin cyanide. The dashed lines represent total phosphate concen- tration determined by the method of Ames DPG concentration.

gible amounts of organic phosphates can be removed from hemolyzates by gel filtra- tion in the course of 144 hr provided their pH is above neutrality. By contrast, at lower pH variable and significant amounts of phosphate are retained (Table I). It is, therefore, likely that the significant amount,s of DPG occasionally encountered by some workers in hemoglobin solutions after gel filtration (5) resulted from the use of hemolyzates wit’h too low a pH. Several factors, notably the age of the blood and the nature of the anticoagulant can lead to hemolyzates with a pH well below neu- trality. This is part,icularly so with blood collected in acid-citrate-dextrose (ACD), which is commonly used in blood banks. In this laboratory neutral citrate has al- ways been employed as an anticoagulant, as recommended by Drabkin (9).

The correlation between the pH of the hemolyzate and retention of organic phos- phates also holds when dialysis instead of gel filtration is used for the separation, as shown in Table II. This supports the inter- pretation that the increasingly positive charge on t,he protein wit’h decreasing pH is responsible for greater electrostatic binding of the organic phosphate poly- anions. Above neutrality, on the other hand, where the interaction between the phosphate ester and t’he protein is sue- ciently weakened, a complete separation can be achieved.

_ _ and Dubin i6). This is, of course, twice the

TABLE I

PHOSPHATK CONTENT OF HEMOLYZ.\TES AFTER

GEL FII,TR.~TION~

Hemolyzate PH Phosphate content

of eluted Hb (moles P/mole Hb)

J. 0. 7.5 0.03 J. 0. 6.5 0.32 M. B. 7.5 0.04 M. B. 6.5 0.38 s. R. 7.5 0.04 S. R. 6.5 0.28 H. C. 7.5 0.06 H. C. 6.5 0.41 D. S. 7.5 0.04 D. S. 6.5 0.65

a Hemolyzates were prepared from washed

erythrocytes by freezing and thawing followed by treatment with an equal volume of a 10% suspension of DEAE-cellulose in 3 ml phosphate

buffer, pH 7.0, then centrifuged at 45,oOOg for 20 min (8). After adjustment to the desired pH, the hemolyzates were recentrifuged in order to

obtain absolutely clear hemoglobin solutions. This is essential for successful chromatographic separation. The conditions for gel filtration were

the same as those described in Fig. 1. The hemo- globin solutions applied to the column (approxi- mately 2 X 1O-3 M) are diluted about 3-fold by

passage through the column, and the hemoglobin collected and analyzed for phosphate (6) repre- sents about 95$& of the total.

It is, however, striking that, the mobility of DPG itself is much greater than would be expected from its molecular weight (Fig. 2).

238 BERMAN, BENESCH, AND BENESCH

DPG (mol wt 266) evidently has the same mobility as bacitracin (mol wt 1411).

Reversible aggregation can be ruled out as an explanation for this phenomenon, since the mobility remained constant over a 25- fold concentration range (Fig. 2). It was, therefore, suspected that the high mobility of DPG is related to an enlarged Stoke’s radius caused by electrostatic repulsion between the negative charges, giving the molecule an extended conformation. There- fore, protonation of these charges should result in a collapse of the molecule, and a

TABLE II

PHOSPHATE CONTENT OF HEMOLYZATES

AFTER DI.~LYSIS~

Hemolyzate PH Phosphate content of dialyzed hemolyzate (moles P/mole Hb)

1 7.5 0.06 6.5 0.60

2 7.5 0.05

6.5 0.20

3 7.5 0.04

6.5 0.32

(1 Hemolyzates were prepared as described in Table I. After adjustment to the indicated pH,

3-ml aliquots were dialyzed as described previ-

ously (4) and analyzed for phosphate (6). 1.6 1.4

1.2

Pi

Elution Volume [ml)

FIQ. 2. Mobility of DPG on Sephadex G-25. The column (1.5 X 52 cm) was calibrated with blue Dextran (B.D.) for Vo and inorgnic phos-

phate (Pi) for V, Sample volumes were 5 ml. Filled circles, 1 X 1OW M DPG, open circles, 2.5 X 10-a M DPG. Dashed line, bacitracin, 2 mg/ml. The column was equilibrated with 0.1 M NaCl and eluted with the same solvent at 15 ml/hr at +3”. Blue Dextran was measured at 625 rnp, bacitracin at 280 rn$, and DPG and inorganic phosphate by the method of Ames and Dubin (6).

2.3.DPG Pi

FIG. 3. Effect of pH on the mobility of DPG on Sephadex G-25. The column is described in the

legend to Fig. 2. It was equilibrated and eluted with the following solutions : pH 2.06, 0.01 M HCl-

0.1 M NaCl; pH 46, 0.01 M sodium acetate buffer- 0.1 M NaCl; pH 8.11,O.Ol M Tris buffera. M NaCl. Five milliliters of 7.5 X l(ra M DPG or 15 X lo+ M

inorganic phosphate in 1 M NaCl were used to charge the column.

104- 0 2102-

SIOO- 90 22 98. B : 96- .a ; 94-

1 :: 50

92 f-

2 4 6 a PH

FIG. 4. Comparison of the Ionization of DPG

with its Mobility on Sephadex. Dashed line, titration curve (4) Solid line, elution volume from

the data in Fig. 3.

decrease in its mobility on Sephadex. The effect of pH on the rate of gel filtration of DPG is shown in Fig. 3. It is evident that there is indeed a large decrease in mobility when the pH is lowered from 6 to 2, whereas no change is observed between pH 6 and 8. By contrast, the mobility of inorganic phos- phate changes only very little between pH 6 and 2. From a comparison of the titration curve of DPG with its rate of gel filtration (Fig. 4) it appears that ionization of three groups is sufficient to transform the molecule into its fully extended state. In addition, molecules of hydration can be

REMOVAL OF ORGANIC PHOSPHATES FROM HEMOGLOBIN 239

expected to contribute furt,her to the effec- tive size of the polyanion.

The results presented here show that hemoglobin can be prepared essentially free of organic phosphate either by gel filtration or dialysis provided the solution is adjusted to pH 7.5 and 0.1 M KaCl is present. It must be borne in mind, how- ever, that the intrinsic mobility of DPG at this pH is anomalously high, so that an appropriate column volume is essent.ial for satisfactory separaGons.

ACKNOWLEDGMENTS

The authors thank Dr. Arthur Rowe of t,he New York Blood Center for generous gifts of fresh

blood.

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AND GRISOLI~, S., Fed. Eur. Biol. Sot. Lett.

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8. OSKI, F. A., GOTTLIW, A. J., MILLER, W. W., AND 1>~~LIVORI.4-P.4PPADOPOULOS, M., J. Clin. Invest. 49, 400, 1970.

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