Examination of magnesium binding to serum proteins by ultracentrifugal analysis

ANALYTICAL

BIOCHEMISTRY

Examination

22,

168-116

(1968)

of Magnesium

Binding

Ultracentrifugal NORMAN

A. CUMMINGS,’ HERBERT

Laboratory of Biochemistry, National of Health, U. S. Public Health Education and Welfare, Received

to Serum

Proteins

by

Analysis EDWARD A. SOBER Cancer Service, Bethesda, May

Institute, Department Maryland

L. KUFF,

AND

National Institutes of Health, 2001&

1, 1967

Magnesium ion is of great physiological interest, particularly as a cofactor in many important enzymic reactions. It seems usually to be associated with proteins in loosely bound complexes rather than in the form of metalloproteins per se (1). This loose binding makes it difficult to determine the distribution of magnesium among the individual components of protein mixtures, since buffers and other materials used in fractionation procedures may serve to displace the metal from its original binding site(s). For example, Himmelhoch et al. (2) found the serum magnesium to emerge with the unretarded 75 y-globulin fraction when dialyzed serum was chromatographed on DEAE-cellulose in a tris-succinate buffer system; however, they could not regard this as evidence for a true magnesium-protein association because further dialysis against buffer removed the magnesium from the y-globulin fraction. Furthermore, magnesium chloride, when applied to the adsorbent under the same conditions but in the absence of protein, emerged in the same position. Other experiments with column electrophoresis and gel filtration on Sephadex G-100 both carried out in the presence of tris-succinate buffer, resulted in essentially complete separation of serum magnesium from the protein components. None of these methods, therefore could provide information as to any possible protein binding of serum magnesium. Evidence for loose binding of magnesiums to serum was also obtained from controlled dialysis experiments in which all magnesium was found to be dialyzable, although the rate of escape of serum magnesium from the dialysis sac was somewhat retarded in comparison to MgCl, itself. Both low pH and high conductivity facilitated magnesium escape. ‘Present address: National Health, Bethesda, Maryland

Institute 20014.

of Dental 108

Research,

National

Institutes

of

MAGNESIUM

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In the present paper we describe results with serum from the use of a preparative sedimentation technique in a swinging-bucket rotor, and indicate how this method may be used to study the distribution of serum components (e.g., magnesium) free of the possibly competitive or inhibitive effects of buffer systems or chromatographic materials. MATERIALS

AND

METHODS

In general, materials were handled as described by Thiers (3) and Himmelhoch et al. (2) in order to free apparatus and buffers from metallic ions, and to prevent contamination of samples and standards. Serum was collected under “metal-free conditions.” Measurement of magnesium was carried out by atomic absorption spectrophotometry (see 2, 4). In our hands a linear standard curve was obtained from 0.01 to 1.0 pg/ml. No interference was noted here with the buffers used. The greatest error in measurement was 1.26%. (For a further evaluation of the accuracy of these methods, see Wacker et $. (5) .) Polyacrylamide gel disc electrophoresis was performed in a Canalco electrophoresis apparatus in 1 M tris-hydrochloride, pH 8.6. Ultracentrifugal analyses were carried out by a modification of the method of Hogeboom and Kuff (7) in a swinging-bucket (SW-39) rotor in either a model E analytical or a model L preparative ultracentrifuge (Beckman Instruments, Inc.). Cellulose nitrate centrifuge tubes (0.5 X 1.2 in.) were filled with a total of 4.6 ml of the serum sample, added as two equal aliquots, the first of which contained 3y0 metal-free sucrose. Gentle swirling of the tubes resulted in partial mixing of the two aliquots and the establishment of a sucrose gradient which subsequently helped to stabilize the protein boundaries against convective disturbances during centrifugation and deceleration of the rotor. Early experiments employed the model E analytical ultracentrifuge, which permits rather close control of rotor speed during deceleration. However, identical results were later obtained when centrifugation was carried out in the model L ultracentrifuge, and the rotor simply allowed to decelerate without braking. Sampling was performed by puncturing the bottom of the centrifuge tubes and collecting the effluent in tared vessels. ilfter weighing, the samples (between 15 and 25 per centrifuge tube) were diluted with a known quantity of buffer before analysis of magnesium and optical density. The analytical results were corrected for dilution (using a value of 1.039 gm/ml as an average density for serum), and expressed as fractions of the original serum concentrations of magnesium (in pg/ml) or of protein (in OD 280 rn,p units/ml). These fractional concentrations (d&gnated CF magnesium or CF protein, respectively), were then plotted

110 against

CUMMINGS,

the distance

KUFF,

AND

SOBER

samples from the meniscus to patterns shown in Figures 1 through 4.

of the respective

obtain the sedimentation

Ultracentrifugation

An analysis of normal undialyzed human serum is shown in Figure 1. The protein distribution (C, protein) revealed two main sedimentation boundaries (arrows, Fig. 1). In addition, a band of absorbing material *MAGNESIUM

I DISTANCE

FROM

2 MENISCUS

3 (cm. 1

FIQ. 1. Sedimentationanalysisof normal undialyeedserumperformedin a model E ultracentrifuge at. 39,460rpm for 12 hr at 20°C: (‘0) CPprotein, (0) CRmagnesium. Meniscus is at left,.

was present at the meniscus, and a large amount of had collected at the bottom of the tube. Calculated of the two boundaries were approximately 4S and they represented primarily albumin and r-globulin. was confirmed by polyacrylamide

sedimented protein sedimentation rates 75, suggesting that This identification

disc gel electrophoresis of the gradient

fractions. The meniscus band, which grossly had a yellow lipoidal of P1-globulin @-lipoprotein) on appearance, consisted mainly electrophoresis.

MAGNESIUM

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PROTEINS

About 30% of the total serum magnesium appeared to have moved as a single sedimenting boundary, corresponding in position to the albumin boundary; the remainder of the magnesium did not. appear to sediment appreciably. No concentration of magnesium appeared in the P-lipoprotein band, nor was there a sedimenting boundary of magnesium associated with the y-globulin. 2.0-

t 0” l.OI

.a f .6 .4 .2 r I I

I DISTANCE

FROM

I

I

2

3

MENISCUS

(cm.)

FIG. 2. Sedimentation a,nalysis of normal dialyzed serum performed in a model L ultracentrifuge at 39,460 rpm for 16 hr at 30°C: (0) CP protein, (0) CR Magnesium. Serum was first dialyzed for 24 hr against a 25-fold volume of 0.04 A4 tris0.005 M succinate, pH 8.6 buffer.

In additional experiments (not illustrated here), normal undialyzed serum was centrifuged at 30” and at 2O“C for times up to 16 hr. In each instance the magnesium boundary was observed to correspond in position with that of serum albumin, and the nonsedimenting fraction represented about 70% of the total magnesium. The effect of dialysis upon the magnesium sedimentation pattern is illustrated in Figure 2. In this case, serum was first dialyzed against tris-succinate starting buffer for 24 hr, and the nonsedimenting magnesium now represented only 1570 of the total. The sedimenting mag-

112

CUMMINGS,

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nesium boundary now appeared significantly displaced from the main protein boundary, having moved 0.2 cm farther from the meniscus. This same phenomenon was observed in the experiment shown in Figure 3. Here the serum was taken from a subject who was subsequently found to have hyper-pre-P-lipoproteinemia (Fredrickson, Type 3)) a metabolic !.5 -

- If

t I I

. MAGNESIU-M 0 PROTEIN A O.D. 460 mp

- !

LO -

I

- I

1

- 1

.8.6 -;

DISTANCE

FROM

MENISCUS

(cm.)

FIG. 3. Sedimentation

analysis of hyperlipemic dialyzed serum performed in a model L ultracentrifuge at 39,460 rpm for 16 hr at 30°C: (0) CP protein, (0) Cr magnesium, (A) OD 460 rnp. Serum was first dialyzed for 24 hr against a 2.5-fold volume of 0.04 M tris-0.005 M succinate, pH 8.6 buffer.

disease of carbohydrate metabolism resulting in high serum p-lipoproteins (8) * An enormous yellow lipoprotein band was present at the meniscus, and an additional yellow band was visible approximately 1 cm from the meniscus. This yellow color, presumably resulting from lipoproteinassociated carotene, was monitored by absorbency at 460 mp (9). The

MAGNESIUM

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main protein sedimentation boundary was in the expected position. Very little nonsedimenting magnesium was detected. The gradient fractions in the above experiment were analyzed by disc gel electrophoresis in order to locate the albumin boundary more pre2.5 MAGNESIUM x”TRANSFERRIN” l

v ALBUMIN

.8 .6

2

I DISTANCE

FROM

MENISCUS

3 (cm.)

FIG. 4. Sedimentation analysis of hyperlipemic dialyzed serum as in Figure 4: (VI CR albumin, (0) CR magnesium, (X) Cs “transferrin”. The CR proteins were calculated on the basis of a scan of disc ael electroohoresis. Proteins are designated according to their electrophoretic mobility and approximate sedimentation rates (see text).

cisely in the presence of the excessive p-lipoprotein. The disc gel patterns, stained with amido black 10-B, were scanned in a Spinco Analytrol with a Spinco No. 300-654 microanalyzer attachment (Beckman Instruments, Inc.) and relative albumin concentrations were determined from the areas under the albumin peak. The plateau in the albumin boundary

114

CUMMINGS,

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SOBER

was arbitrarily set at Cr = 1. Results are shown in Figure 4, which clearly illustrates the displacement of the magnesium ahead of the albumin boundary. In the electrophoretic analysis, a discrete protein band was observed to have a distribution similar to that of magnesium. The relative distribution of this protein, which was not identified here but which showed a sedimentation rate (about 55) and electrophoretic behavior similar to transferrin (lo), is also shown in Figure 4. DISCUSSION

Ultrafiltration studies have indicated that about 35% of serum magnesium is bound to proteins (11). Dialysis experiments have shown that all the magnesium can be removed from serum proteins under proper conditions. Therefore an association of the magnesium with serum proteins must be in a relatively loosely bound form. These facts are in agreement with studies of the binding of magnesium with isolated serum components, which showed the formation of dissociable magnesium and calcium protein complexes in accordance with mass action behavior (12). Since the binding of magnesium to serum proteins is loose and dissociable, it would be expected that examination of the partition of magnesium among the various proteins would be difficult. Thus the conditions used in a number of fractionation procedures, e.g., DEAE-cellulose chromatography, gel filtration, and electrophoresis, resulted in complete displacement of magnesium from the otherwise fractionated serum proteins. The principle of sedimentation analysis to study the behavior of metalprotein complexes was used as long ago as 1942 to examine the partition of protein and metal in solutions of calcium caseinate (13). The assumption is also made in the present work that the diffusible metal remains evenly distributed throughout the fluid phase, while the bound metal is sedimented with the protein. Our sedimentation analysis has resulted in an estimate of the percentage of free and protein-bound magnesium which is in good agreement with the previously mentioned studies. Furthermore, the method has given additional information on the possible proteins involved in the complex: in undialyzed serum, the sedimentation rate of magnesium was the same as that of albumin. This is in line with expectations: Copeland and Sunderman have shown that the ratio of “magnesium-binding power” of isolated albumin to globulin is 1.53 (11). Using purified bovine fractions, Carr and Woods also demonstrated a greater affinity of magnesium for albumin than for globulin (14). As the pH decreased, the percentage of magnesium bound to albumin also diminished (14). However, results must be interpreted with care, since no positive

MAGNESIUM

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identification of the protein involved is possible with sedimentation analysis; the magnesium may be sedimenting at the same rate of speed, but not necessarily associated with, the albumin. Our results have given no evidence for binding of magnesium to serum lipoprotein or y-globulin but it would be difficult to detect the binding of small amounts of magnesium to different proteins. The observation that magnesium sediments at a faster rate in serum dialyzed against tris-succinate is unexplained. One tentative hypothesis is that dialysis resulted in displacement of the weakly bound metal from albumin to a faster sedimenting serum protein. Another possibility is that dialysis changed the physical properties of the magnesium-binding protein. It should be emphasized that the protein designated “transferrin” (Fig. 4) is just one of many possible proteins sedimenting in the area faster than albumin; the ability of centrifugation to resolve proteins of similar sedimentation rates is limited, at the present stage of the technique. Finally, limitations in the general precision of the method must be pointed out. Chances for nonsystematic errors in weighing, dilution fact.ors, convective disturbances during manipulation, and magnesium determination occur, and may be cumulative. Nevertheless, this technique for the study of sedimenting proteins is of value where the distribution of substances involved might be otherwise affected by the presence of buffer systems or other experimental factors. SUMMARY

AND

CONCLUSIONS

1. Sedimentation of serum in a horizontal rotor has been used with metal-free techniques. This method was employed in order to avoid the displacement of magnesium inherent in the other fractionation procedures. Results indicate that 70% of the magnesium in normal human serum is unbound. The rest sediments at the same rate as the albumin. Little or none traveled with p-lipoprotein or y-globulin. 2. In serum dialyzed against tris-succinate, the magnesium sediments faster than albumin but more slowly than y-globulin. The reason for this effect of dialysis is not understood. 3. Results are discussed in relation t,o other studies, and some of the values and limitations of the technique are mentioned. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Mr. Hugh Foster. Dr. Cummings was a Research Associate, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, U. S. Public Health Service, when this work was done.

CUMMINGS,KUFF,AND

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REFERENCES

1. VALLEE, B. L., in “The

Enzymes” (Bayer, P. O., Lardy, H., and Myrbkck, K., 3, Part B, p. 225 Academic Press, New York, 1960. HIMMELHOCH, S. R., SOBER, H. A., VALLEE, B. L., PETERSON, E. A., AND FUWA, K., Biochemistry 5, 2523 (1966). THIEFGS, R. E., Meth. Biochem. Anal. 5,273 (1957). FUWA, K., AND VALLEE, B. L., Anal. Chem. 35, 942 (1963). WAC-, W. E. C., IIDA, C., AND FUWA, K., Nature 202,659 (1964). PETERSON, E. A., AND CHIAZZE, E. A., Arch. Biochem. Biophys. 99, 136 (1962). HOQEBOOM, G. H., AND KUFF, E. L., J. Biol. Chem. 210,733 (1954). FREDRICKSON, D. S., Circulation 31, 321 (editorial) (1965). ONCLEY, J. L., GURD, F. R. N., AND MELIN, M., J. Am. Chem. Sot. 72, 458 (1950). PUTNAM, F. W., in “The Proteins” (4eurath, H., cd.), Vol. 3. 13. 154. Acad:bmic Press, London, New York 1965. COPELAND, B. E., AND SUNDERMAN, F. W., J. Biol. Chem. 197, 331 (1952). CARR, C. W., Proc. Sot. Exptl. Biol. Med. 89, 546 (1955). CHANUTIN, A., LUDEWIG, S., AND MUSKET, A. V.. J. Biol. Chtm. 143, 737 (1942). CARR, C. W., AND WOODS, K.. Arch. Biophvs. Biochem. 55, 1 (1955). eds.),

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