Heterogeneity of human serum high-density lipoprotein (HDL2)

Clinica

Chimica

&JElsevier

Acta,

43

(1973) 223-229

Scientific Publishing

Company,

Amsterdam

- Printed in The Netherlands

223

cc* 5456

HETEROGENEITY

OF HUMAN

LIPOPROTEIN

SERUM

HIGH-DENSITY

(HDL,)

S. L. MACKENZIE&.

G. S. SUNDARAM”

AND H. S. SODHIb

a Prairie

Regional Laboratory, National Research Council Saskatoon, Saskatchewan and b Department of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan (Canada) (Received

August

29, 1972)

SUMMARY

Serum high-density lipoprotein (HDL,) has been shown by preparative isoelectric focusing, to consist of several discrete lipoprotein moieties having different isoelectric points, protein to cholesterol and protein to phospholipid ratios. Recombination of these moieties yielded a product indistinguishable from HDL, on examination by analytical ultracentrifugation and agarose electrophoresis.

INTRODUCTION

The heterogeneity of the protein moiety of serum high-density lipoprotein (HDL) has been well established”3. However, HDL is generally regarded as consisting of a continuum of species varying in molecular weight and hydrated density4v5. The concept of continuity of chemical and physical properties within this class is further supported by homogeneity during free boundary electrophoresise and during electrophoresis on media such as agarose’ and paper. HDL, and HDL, are arbitrary divisions of this continuum. There is relatively little evidence to suggest that the variation in properties may be discrete although HDL and its sub-classes, HDL, and HDL,, have been separated into several components by electrophoresis on starch gel* and polyacrylamide ge18. Albers and Aladjemlo have also presented immunochemical evidence suggesting the existence of two HDL populations having different polypeptide compositions lo. A previous study, using analytical isoelectric focusing, on serum lipoproteins which had not been purified, suggested the possibility of the existence of discrete subclasses in HDLrr. Our studies with purified lipoproteins demonstrate that HDL, may, in fact, be separated into several discrete lipoprotein fractions by preparative isoelectric focusing. MATERIALS

density

AND METHODS

HDL, was prepared from fresh plasma by ultracentrifugal flotation of lowlipoproteins at a density of 1.065 g/ml followed by flotation of HDL, at

Issued as NRCC No. 12944 For reprints contact Dr. H. S. Sodhi.

MACKENZIE et al.

224

1.125 g/ml. Solid NaBr was used to raise the density of the solution and ultra-centrifugation was performed in a Beckman Spinco Model Lz-65B preparative ultra-centrifuge using a 60 Ti rotor atz5oooo x g for 24 h at 16”.The HDL, was washed twice in a NaCl solution of density 1.125 g/ml and dialysed against 0.15 M NaCl, pH 7 containing 0.05% EDTA at 4’. HDL, was labelled with lZsI by the method of McFarlane 12.Immediately prior to electrofocusing, the lipoprotein solution was dialysed against distilled water. Cholesterol was determined by the method of Abel1 et al.13 and phospholipid by the method of Bartlett 14. Polyacrylamide gel electrophoresis was performed as previously describedI except that the acrylamide composition was 12% and that no urea was used. Isoelectric focusing was performedI at IO’ using 2% carrier ampholytes (Ampholine, LKB-Produkter AB, Stockholm) and an LKB, Model 8101, electrofocusing column. Solutions and density gradients were prepared as described by LKB Instruments17. The initial voltage and current ranged from 250-350 V and s-10 mA to give the desired applied power of 2.5-X watts. The power was maintained at this level throughout each experiment and the resultant final voltage and current ranged from 500-600 V and 4-6 mA respectively. The precise voltage and current depended on the amount of sample applied. Equilibrium was usually reached in 48 h. The protein load varied from 50 to 200 mg. Initially a pH range from 3 to IO was used. However, in agreement with the analytical findings of Kostner et al.11, it was observed that HDL focused in the range 3 to 6. This range was, therefore, used for all subsequent studies. Fractions of 1.5-2 ml were collected. The pH of each fraction was determined at 25” using a Sargent-Welch, Model NX, pH meter. The protein content of individual fractions was determined by measuring the absorbance density at 280 nm in a Beckman Model DB-G spectrophotometer and by the method of Lowry et al.ls after Ioo-fold dilution to minimize interference from the Ampholinesla.

RESULTS

AND

DISCUSSION

Typical results obtained by isoelectric focusing of HDL, are shown in Fig. I. The absorbance curve indicates the presence of discrete fractions. Differences in the isoelectric points of the peaks were observed with different samples and may be due to inherent differences in the sera 11~20. However, as shown in Table I, most of the TABLE

I

ISOELECTRIC

POINTS

OF HIGH

DENSITY

LIPOPROTEIN

.%Wlp1e A B C D E

(HDL,)

FRACTIONS

pr values

3.80 _* 3.72 3.75 3.90

4.15 4.05 4.05 4.05 4.10

.

4.34 4.32 4.37 4.38 4.35

4.70 4.58 4.84 4.55 -

5.00 5.02 5.01.

5.45 5.42. .j.12*

j.01

5.02

5.46 5.45 5.45

5.66*

6.70 6.07 6.70 6.62 6.54

Values represent the p1 of peaks or shoulders in five different experiments using different preparations of HDL,. The same HDL, preparation was used for experiments D and E but different amounts of sample were applied. The component of p1, approximately 4.5, was also present in sample E but not in sufficient quantity for its isoelectric point to be determined. Samples A and B were iodinated; samples C, D and E were not. * These components

were either missing or present only in some samples.

HETEROGENEITY

OF HDL,

FRACTION

225

NUMBER

Fig. I. Isoelectric focusing of HDL, labelled with lesI. d denotes pH, o absorbance 0 counts per minute per fraction.

at 280 nm and

lipoprotein fractions consistently focused in a narrow pH range. The protein distribution in individual fractions closely paralleled the absorbance curve. A narrow flocculent zone, corresponding to the shoulder at about pH 4 and remaining in the same location throughout the experiment was observed in some experiments. This precipitate was readily solubilized on dilution. Other than this, complete solubility was maintained throughout the experiment. Precipitation could be eliminated by applying a smaller sample. The distribution of protein, cholesterol and phospholipid in pooled fractions is shown in Table II. These data represent the mean values from two experiments using the same HDL, sample. Cholesterol and phospholipid were present in each pool TABLE

II

DISTRIBUTION (%

Pooled peak* * I

2 3 4 5

OF PROTEIN,

CHOLESTEROL

AND

PHOSPHOLIPIDS

IN

HDL,

FRACTIONS

of total amount recovered*) PI values D E D E D E D

3.75. 4.05 3.90, 4.10 4.38, 4.55 4.35, -

E

5.45

D

6.62

5.01

5.02 5.45

o/OProtein

0/OCholesterol

0/oPhospholipid

4.5 i

0.6

9.7 + 7.0

I.4 zt 0.4

17.5 f

0.9

8.9 & 0.I

4.5 i

54.2 f

0.3

64.2 + 9.0

72.9 & 7.7

15.4

3

2.6

14.3 i

17.1

8.5

*

3.2

2.9

i

2.4 0.6

*

0.8

5.0

4.2 + 1.3

E 6.54 Values represent the mean of two determinations. * The amounts recovered, expressed as o/0 of the amount applied were protein (96.6 & 3), cholesterol (94 -& 4) and phospholipid (94 f 3). ** A pool was formed by combining individual fractions constituting a protein peak with a characteristic p1 value. In some cases fractions of more than one p1 value were pooled since they represented adjacent shoulders.

226

MACKENZIE

et al.

and were assumed to be combined with protein. The peaks with different isoelectric points therefore represented lipoprotein species. The differences in the protein: cholesterol and protein:phospholipid ratios of the pooled fractions, although not conclusive, suggest the existence of different lipoprotein species in HDL,. Since the various components were not completely resolved, the compositions given in Table II are presented only to support the suggestion of heterogeneity and do not represent the compositions of the individual components. The results of experiments with 1251-labelled HDL, (Fig. x) showed that the specific activity of the peptides in different fractions was not the same, demonstrating heterogeneity in the distribution of the proteins available for iodination. The pooled fractions containing peptides of highest specific activity also had highest lipid: protein ratios (Table II). This agrees with the observations of Sundaram et ~Z.~rthat delipidation of iodinated HDL yielded peptides of different specific activity and that proteins with highest specific activity had greater affinity for lipids. Polyacrylamide gel electrophoresis of total HDL, and the fractions obtained by isoelectric focusing (Fig. 2) provide further evidence for heterogeneity in the distribution of proteins.

-~ + HDL2

! I

2

3

4

5

Fig. 2. Polyacrylamide gel electrophoresis patterns. The numbers correspond to the pooled fractions indicated in Table II.

There are distinct qualitative and quantitative differences in the protein composition of the various fractions. Recombination of fractions and subsequent study by analytical ultracentrifugation and agarose gel electrophoresis 7 demonstrated the presence respectively of a single peak and a single lipid staining band indistinguishable from the starting material. The Schlieren pattern obtained for recombined HDL, is shown in Fig. 3. The sedimentation coefficients of the original and recombined materials were respectively, 4.20 and 4.24 S. The agarose gel electrophorogram of HDL, and recombined HDL, fractions is shown in Fig. 4. The presence of Ampholine, in the same concentration as that used for electrofocusing, had no effect on the electrophoretic mobility. These results suggest that the lipoproteins have not undergone significant irreversible denaturation during isoelectric focusing. The possibility that the observed fractionation is artefactual must be considered. In studies on acidic proteins the formation of artefacts appears to depend on the presence of and interaction with basic Ampholineszz. In the present case, only the

HETEROGENEITY

OF HDL,

227

Fig. 3. Sedimentation pattern of HDL,. 4 mg protein/ml w‘ere dissolved in 0.05 M carbon atebicarbonate buffer, pH 9.6. The original and recombined lipo ‘proteins are shown in the lower and upper image respectively. Operating conditions were 60000 rev./min, 2o”, analyser angle 50”. The photograph was taken after 36 min at top speed.

Fig. 4. Agarose gel electrophoresis (right hand image).

of HDL,

(left hand image) and recombined

HDL,

fractions

MACKENZIE

228

t?t al.

pH range 3-6 was used but even in the preliminary experiments using a 3-10 pH range comparable results were obtained. Furthermore, in experiments using 3Hlabelled Ampholine, Hayes and Wellner a3demonstrated that little, if any, Ampholine was bound to protein and that heterogeneity was due only to differences in the primary structure of the proteins separated. Although LDL and VLDL readily lose lipid, HDL is considerably more stable. Notwithstanding that it is generally accepted that lipoproteins are unstable at their isoelectric points, our results indicate that HDL, is, in fact, quite stable under the conditions used for isoelectric focusing. In studies using Ampholine carrier ampholytes for isoelectric focusing on polyacrylamide gels, Righetti and Drysdalez4 observed that several absorbance peaks could be ascribed to the Ampholine chemicals. These peaks and the general background absorbance created by the Ampholines (A:;$ < 0.03) are significant in analytical situations where the protein load is small2Obut are of no significance in preparative studies. We have verified in a control experiment containing no sample that there were no peaks (A at 280 nm) in the relevant range. Furthermore, there were no significant differences in the position or proportions of the peaks when different amounts of the same sample were subjected to isoelectric focusing. Iodinated and non-iodinated preparations yielded generally similar results, The heterogeneity observed, therefore, could not have been an artefact of the iodination process. Although the possibility of artefact formation has not been completely eliminated, the evidence does suggest that there was no interaction between HDL, and Ampholine and that the iodination process was not responsible for the heterogeneity observed. Since the recombined lipoprotein fractions constituted a product indistinguishable from the starting material, it seems reasonable to conclude that isoelectric focusing did not degrade or denature the HDL, and that the observed results suggest that HDL, consists of several quite discrete lipoprotein fractions. ACKNOWLEDGMENT

This work was supported in part by the Canadian and Saskatchewan Foundations and by the Medical Research Council of Canada.

Heart

REFERENCES

SIIORE AND V. SHORE, Biochemistry, 8 (1969) 4510. M. SCANU, J. TOTH, C. EDELSTEIN, S. KOGA AND E. STILLER, Biochemistry, 8 (1969) 3309. J. ALBERS, L. V. ALBERS AND F. ALADJEM, Biochem. Med., 5 (1971) 48. L. ONCLEY, Biopolymers, 7 (1969) 119. A. M. SCANU, in E. TRIA AND A. M. SCANU, Structural and Functional Aspects of Lipoproteins

I R. A. J. J.

2 3 4 5

6 7 8 9 IO

II 12 13

14 15 16 17

in Living Sysfems, Academic Press,New York, 1969, Chapter C3. B. SHORE AND V. SHORE, Biochem. Biophys. Res. Commun., 1 (1959) 228. R. P. NOBLE, J. Lipid Res., g (1968) 693. A. M. SCANU AND J. L. GRANDA, Progr. Clin. Pathol., I (1966) 398. K. NARAYAN,S.NARAYANAND F. A. KUMMEROW, Nature, 205 (1965) 246. J. J. ALBERS AND F. ALADJEM, Biochemistry, 10 (1971) 3436. G. KOSTNER,~. ALBERT AND A.HOLASEK, Hoppe-Seyler’sZ.Physiol. Chem., 350(1969) 1347. A. S. MCFARLANE, Nature, 182 (1958) 53. L. L. ABELL, B. B. LEVY, B. B. BRODIE AND E. J. KENDALL, J.Biol. Chem., 195 (1952) 357. G. R. BARTLETT, J. BioZ. Chem., 234 (1959) 466. H. S. SODHI, R. K. BHATNAGAR AND S. L. MACKENZIE, Can. J. Biochem., 4g(Ig7r) 1076. 0. VESTERBERGAND H. SVENSSON, Acta Chem. &and., 20(1g66) 820. Instruction Manual, LKB-Produkter AB, Stockholm-Bromma I, Sweden

HETEROGENEITY

229

OF HDLz

18 0. H. LowRY,N.J. ROSEBROUGH, A.L.FARRAND 265. 19 M. PAGE, Biochim. Biophys. Acta, 236 (1971) 571.

R.J.

RANDALL, J.Biol.

Chem.,

20 C. RITTNER AND B. RITTNER, 2. Klin. Chem. Klin. Biochem., g (1971) 503. G. S. SLJNDARAM, H. S. SODHI AND R. BHATNAGAR, Clin.Chim. Acta, 3g(Ig72) 22 R. FRATER, J. Chromatog., 50 (1970) 469. 23 M. G. HAYES AND D. J. WELLNER, J. Biol. Chem., 244 (1969) 6636. 24 P. RIGHETTI AND J, W. DRYSDALE, Biochim. Biophys. Acta. 236 (1971) 17. 21

Igj(Ig65)

115