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Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins
ANALYTICAL
BIOCHEMISTRY
88,
425-433 (1978)
Preparative Isoelectric Focusing of Human Serum Low-Density and Low-Density Lipoproteins K.M.
SHANMUGAG.SUNDARAM,
MOHAMED~HAKIR,AND MARGOLIS
SIMEON Departments
of Medicine
Very
and Physiological Medicine, Baltimore,
Chemistry, Maryland
The Johns 21205
Hopkins
School
oj
Received June 1, 1977; accepted March 17, 1978 Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF). IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [W]Ampholine, and gel filtration completely separated the lipoproteins from [W]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.
Several workers have used isoelectric focusing (IEF)’ to study the apoproteins of very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Blaton and Peeters (1) separated human apo-LDL into a major component with a pZ value of 5.36 and a minor one with a pZ value of 6.12, both unreactive with antisera against human LDL. They concluded that apoprotein B consists of two different polypeptides (2). Albers and Scanu (3) used the IEF technique to separate human apo-VLDL into four different polypeptides, each with distinct pZ values and chemical properties. However, when IEF was used to study intact lipoproteins and partially delipidated lipoproteins obtained by extraction with organic solvents, the lipoproteins and apoproteins tended to precipitate at their isoelectric points. These results led Scanu et al. (4) to suggest that IEF has limited value as a preparative technique for intact lipoproteins. On the other hand, Pearlstein and Aladjem (5) successfully applied the preparative technique to the study of intact VLDL in the presence of 33% ethylene glycol. They showed the presence of five distinct subpopulations of particles, one of which was not reactive with anti-LDL antiserum. We have previously shown (6-9) that use of high concentrations of ’ Abbreviations used: VLDL, very low-density lipoproteins; LDL, low-density lipoproteins; HDL, high-density lipoproteins; pl, isoelectric points; IEF, isoelectric focusing. 425
0003-2697/78/0882-0425$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
426
ST WDARAM,
SHAKIR,
AND
MARGOLIS
Ampholine prevents precipitation during preparative IEF studies of intact human serum high-density lipoprotein (HDL) and its subfractions HDLz and HDL3 and that the separated subspecies are not artifacts. When the same technique was applied to the study of intact VLDL and LDL, precipitation and nonreproducible resolution profiles invariably resulted. Neither 33% ethylene glycol nor other stabilizing agents, except glycerol, completely preventrId the precipitation. In this paper, we describe an adaptation of the preparative IEF technique that gives reproducible separation profiles for intact VLDL and LDL. We also present evidence that the observed optical density peaks are not artifacts arising from interactions between the lipoproteins and the Ampholines used for electrofocusing. Finally, we have shown that preparative IEF can be used to isolate different proteolysis-resistant products of LDL digestion. EXPERIMENTAL
PROCEDURE
Materials. [14C]Ampholine, pH 3- 10 (carboxylic groups labeled; specific activity, 5 &i/g), and Ampholine, pH 4-6, were obtained from LKBProdukter AB, S-161 25 Bromma 1, Sweden. Chloramphenicol was obtained from Parke-Davis Co. Triton X-100 and immobilized Pronase were obtained from J. T. Baker Chemical Co. and Sigma Chemical Co., respectively. All other chemicals and solvents were of reagent grade. Preparation of VLDL and LDL. Two- to three-day-old human serum was pooled from two donors to the blood bank of the Johns Hopkins Hospital. After sodium azide (0.05%), chloramphenicol (50 pg/ml), and EDTA (1 mM) wertr added, chylomicrons were removed by centrifugation at 10,000 rpm in a Ti-50 rotor for 30 min in a Beckman Model L2-50 preparative ultracentrifuge. Subsequently, the chylomicron-free serum was centrifuged at 40,000 rpm for 18 hr in a Ti-50 rotor, and the VLDL fraction was removed by tube slicing. VLDL was purified by two subsequent ultracentrifugal washings under similar conditions with a large excess of 0.85% NaCl-0.05% EDTA solution (PH 7.4). The VLDL-free serum was raised to a density of 1.019 g/ml with solid NaBr, and the intermediate density lipoproteins were removed by centrifugation as described above, The infranatant was centrifuged once again under similar conditions to remove completely any contaminating intermediate density lipoproteins. The density of the infranatant was then raised to 1.05 g/ml with NaBr, and the LDL in the density range 1.019- 1.05 g/ml was floated by centrifugation at 45,000 rpm for 24 hr. LDL was purified by two subsequent ultracentrifugal flotations using salt solutions of density 1.05 g/ml. The purified LDL was dialyzed against 0.85% NaCl-0.05% EDTA solution (pH 7.4) at 4°C and stored in the dark in sealed vials under N2 at 4°C until use. Isoelectricfocusing. Isoelectric focusing was performed at 4°C using 2%
ISOELECTRIC
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carrier ampholytes (pH 4-6) and an LKB Model 8101 electrofocusing column as described previously (7). Initially, solutions and density gradients were prepared as described by LKB Instrument Co. (10) without the addition of any stabilizing agent. In later experiments, distilled water used for the preparation of density gradient solutions was replaced by solutions containing ethylene glycol (10, 20, 33, or 40%), Triton X-100 (O.OS-0.5%) (ll), Triton X-100 (0.05%) and glycerol (5%), or glycerol (10%). Applied samples of VLDL or LDL also contained the same amount of stabilizing agent. In experiments using [‘*C]Ampholine, a final concentration of 2% Ampholine was prepared by the addition of 4 ml of unlabeled Ampholine (pH 4-6) to 1 ml of [14C]Ampholine (pH 3- 10). VLDL or LDL, containing 1 mg of protein as determined by the method of Lowry ST al. (12), was applied to the column. The initial and final voltages and currents were 400 V and 2.5 mA and 800 V and 2 mA, respectively, for both VLDL and LDL. Equilibrium was usually reached hi tween 48 and 60 hr, by which time the pH gradient and protein positions had stabilized. The initial and final voltages varied with the stabilizing agent present, but a constant power of 1.5 to 2.5 W was applied. After each run, approximately 80 fractions (1.5 ml) were collected. The pH and absorbance at 280 nm of the fractions were determined at room temperature and in the presence of sucrose using a Fisher Model 325 pH meter and a Beckman Model DU-2 spectrophotometer. Measurements were made immediately after the fractions were collected to minimize intera,.:Lion of atmospheric COz with the fractions. Gel$filtrution. After exposure to [14C]Ampholine for 24 hr at 4”C, VLDL or LDL was chromatographed on Sephadex G-50 or LKB Ultragel AcA 34 columns (1.6 x 40 cm); 2-ml fractions were collected. Optical density of each fraction was determined as described above. Radioactivity in aliquots of these fractions and in fractions from electrofocusing experiments in the presence of [‘4C]Ampholine was determined using a Beckman Model LS 3150 P scintillation spectrometer. Proteolysis of LDL. LDL in phosphate buffer (1.1 M, pH 7.6) was digested with 1% immobilized Pronase at 37°C with gentle shaking for 20 hr. After the Pronase was removed by two centrifugations at 3000 rpm for 30 min, the clear supernatant was dialyzed overnight against three changes of a solution of 0.85% NaCl-0.05% EDTA (pH 7.4) and subjected to preparative IEF in the presence of 10% glycerol as described above. RESULTS
AND DISCUSSION
In the absence of any stabilizing agent, preparative IEF of intact VLDL or LDL resulted in the formation of one or mo;. llocculant rings in the column. The degree of flocculation increased in proportion to the amount of sample applied. The flocculation or “precipitation” was re-
428
SUNDARAM,
SHAKIR,
AND
MARGOLIS
versible. The “precipitate” eluted from the column as an opaque solution that could not be made optically clear by filtration or centrifugation at 2000 rpm for 2 hr. When diluted with an equal volume of 0.05 M carbonatebicarbonate buffer (pH 9.6), the solution became optically clear. The formation of one or more opaque rings was attributed to the accumulation of aggregated VLDL or LDL particles at their isoelectric points. It was possible to minimize this “precipitation” and to obtain fairly reproducible optical density profiles of the eluted fractions by using 0.5 mg or less of VLDL or LDL protein. Methods that lead to precipitation are undesirable since some of the “precipitate” may stick to the column, sediment or float, and elute with later fractions. As a consequence, the optical density profile of the lipoprotein will vary from one experiment to another. The irreproducible results obtained in our early experiments with VLDL and LDL (data not shown) prompted an investigation of stabilizing agents to prevent “precipitation.” The lirst agent tried was 33% ethylene glycol which Pearlstein and Aladjem (5) used for the IEF of VLDL. They dialyzed native VLDL exhaustively against 33% ethylene glycol and added it to the electrofocusing column in a linear sucrose gradient containing 33% ethylene glycol. In our experiments dialysis of either VLDL or LDL against 33% ethylene glycol usually resulted in the deposition of a fine precipitate in the dialysis bag. Our IEF experiments were carried out as outlined by Pearlstein and Aladjem (5), but with a total of 2 to 3 W of power, since it was felt that the 7.5 W apparently used by these authors may denature lipoproteins. Irrespective of whether VLDL dialyzed against 33% ethylene glycol or the clear supernatant obtained by centrifugation of VLDL dialyzed against 33% ethylene glycol was used, “precipitation” was observed within 24 hr. Pearlstein and Aladjem (5) do not mention the appearance of any “precipitate” during IEF of VLDL. The elution profile they reported for native VLDL also suggests that some precipitation might have occurred since most of the lipoprotein eluted in a single peak, comprising tube numbers 60 to 140, with an optical density greater than 2.0. Different concentrations of ethylene glycol (10, 20, or 40%) were not completely effective in preventing the “precipitation.” Triton X-100 (11) (O.OS-0.5%) was equally ineffective. Although the use of Triton X-100 (0.05%) and glycerol (5%) appeared to prevent the precipitation, a faint opaque ring was noted and isoelectric profiles were not reproducible. Glycerol (10%) completely eliminated the “precipitation” (delined as the appearance of a visible flocculant ring) and gave optical density profiles for LDL and VLDL, as shown in Figs. 1 and 2, respectively. In the presence of 10% glycerol, as much as 6 mg of LDL or VLDL protein was applied to the column without any precipitation. Glycerol at 5 and 2.5%
ISOELECTRIC
FOCUSING
429
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1 6
$0.2
I
2
-51 a
0
10
20
30
40 50 60 Tube Number
-2000
-3
-coo
-J
80
70
-4
100
1 -c ‘E B IJ
FIG. 1. Isoelectric focusing profile of low-density lipoprotein in the presence of 10% (w/v) glycerol. (~1 Azso nm; (0) “C activity; (0) pH. The isoelectric protile is typical or those obtained from three different experiments carried out at 4-day intervals using the same preparation of LDL. The isoelectric focusing time was 60 hr. Fractions containing about 1.6 ml (30 drops) were collected.
did not completely prevent precipitation with the 6-mg protein load. It is not known whether 10% or greater concentrations of glycerol will prevent lipoprotein precipitation at higher sample loads. The optical density profiles shown in Figs. 1 and 2 were highly reproducible in three different consecutive experiments (Table 1) when electrofocusing was carried out for 60 hr. Electrofocusing for a longer period (84 and 96 hr) did not alter the optical density profiles or pH proflies, showing that the pH positions occupied by the proteins were true p1
-7 6 I 51 a -4 -3 0
10
20
30
c--rcI 40 50 60 Tube Number
70
c,
80
\. 90
( 100
I 5 1000fj u^
2030
_
FIG. 2. Isoelectric focusing protile of very low-density lipoprotein in the presence of 10% (w/v) glycerol. (x)A,@ “,,,; (0) “C activity; (0) pH. The isoelectric profile is typical of those obtained from two different experiments using the same preparation of VLDL. Fractions containing about 1.3 ml (25 drops) were collected.
430
SUNDARAM,
SHAKIR, TABLE
ISOELECTRIC
Experiment
POINTSOF
AND MARGOLIS 1
LDL AND VLDL FRACTIONS"
Fraction I
Fraction II
LDL A B C
4.10 4.15 4.15
4.50 4.50 4.50
4.60 4.60 4.60
4.98 5.00 4.98
5.22 5.22 5.22
5.30 5.30 5.30
5.35 5.35 5.35
5.45 5.45 5.45
6.10 6.05 6.10
VLDL A B
4.20 4.20
4.40 4.45
4.55 4.55
4.90 4.96
5.05 5.05
5.15 5.15
5.40 5.40
5.45 5.45
6.10 6.10
a The pZ values represent the pH values of fractions containing the peaks shown in Fig. 1 or 2. Recovery of LDL and VLDL from the column ranged from 96 to 100%. The lipoproteins were distributed in the following proportions: LDL, 36% in Fraction I and 64% in Fraction II; VLDL, 73% in Fraction I and 27% in Fraction II.
values. Furthermore, the current drawn from a constant-voltage power supply was at minimum after 60 hr, showing that the electrofocusing was completed by that time (10). There was no further drop in the current applied to the column on continuing the electrofocusing for 6 more hr, indicating that a true steady-state equilibrium was present. Based on the optical density profile shown in Fig. 1, the eluted material from the IEF of LDL was arbitrarily divided into fractions I and II. Figure 2 clearly shows a similar distinct separation of fraction I from fraction II in VLDL. The smaller peaks of VLDL with pZ values between 4.90 and 6.10 were included in fraction II because of their resemblance to peaks in fraction II of LDL. It is apparent from comparing the isoelectric profiles of LDL and VLDL that several peaks with similar pZ values appear in both lipoproteins. The isoelectric profiles of peaks with pZ values 4.10,4.60, and 6.10 in LDL were strikingly similar to those of peaks with pZ values 4.20, 4.55, and 6.10 in VLDL. The magnitude of most peaks in LDL is greater than the magnitude of peaks with similar pZ values in VLDL. Several prominent peaks in LDL have no counterpart (for example, pZ 5.33) or very small peaks (pZ 5.4) in VLDL. Both LDL and VLDL have some peaks that are not fully resolved from one another. Further electrofocusing experiments using a narrower-pH range Ampholine may allow complete resolution of these peaks. Table 1 shows the excellent reproducibility of the pZ values in different experiments using the same preparation of LDL or VLDL. Neither the resolution pattern nor the pH values of the peaks varied when the experiments were carried out under identical conditions at 4-day intervals. Even though several peaks with different pZ values were observed in the iso-
ISOELECTRIC
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OF LIPOPROTEINS
electric profiles of HDL, HDL*, and HDL3 (6-9), the IEF profiles of highdensity lipoproteins are quite different from those of LDL and VLDL. Considering the heterogeneity of VLDL particles, the presence of several isoelectrically different species is not surprising. However, the apparent isoelectric heterogeneity of LDL was unexpected since LDL is generally considered to consist of rather homogenous particles. The isoelectric heterogeneity of LDL and VLDL suggest that, when these lipoproteins are isolated over a particular density range by ultracentrifugation, they may consist of several subspecies which differ in specific apoprotein content, lipid/protein ratios, or sialic acid or other carbohydrate components. Purification and characterization of each peak is necessary to understand the significance of the observed heterogeneity. However, any hypothesis about the structure of VLDL and LDL must take into account and explain the heterogeneous behavior of these particles on IEF. Many sources of artifact in IEF have been considered (6-9,13). Of greatest concern, however, are possible interactions between Ampholine and the lipoproteins. Therefore, we added labeled Ampholine to lowdensity lipoproteins in experiments similar to our earlier work with highdensity lipoproteins (9). Figures 1 and 2 show the results of electrofocusing of LDL and VLDL in the presence of [14C]Ampholine. Since no 14C activity was eluted with the peaks containing the lipoproteins, it is clear that Ampholine did not bind to either lipoprotein under these conditions of electrofocusing During gel filtration on Sephadex G-50 A.
.60 -
- 6,000 E -5,000 - 4,000
Tube
FIG. 3. Chromatography 2% (w/v) [Y]AmphoIine,
‘E 4 2
Number
on Sephadex G-50 of (A) LDL and (B) VLDL pH 3- 10. (0) A,,, “,,,; (0) 14C activity.
solutions
containing
432
SUNDARAM,
SHAKIR,
AND MARGOLIS
40
Tube
50
60
70
Number
FIG. 4. Isoelectric focusing profile of Pronase-digested LDL in the presence of 10% (w/v) glycerol. (0) AZsO“,,,. Fractions containing about 1.6 ml (30 drops) were collected.
columns both VLDL and LDL eluted with the void volume, whereas all of the labeled Ampholine eluted in a late peak (Fig. 3). We also employed LKB Ultragel AcA 34, a mixture of agarose and polyacrylamide, to examine lipoprotein-Ampholine interactions during gel filtration on a gel other than Sephadex. No significant 14C activity was eluted with the lipoprotein peaks (data not shown). The failure of Ampholine to bind to the lipoproteins in any of these experiments strongly suggests that the heterogeneity observed during electrofocusing is not due to lipoprotein-Ampholine interactions. When LDL was digested with insoluble Pronase and then subjected to IEF, the observed profile (Fig. 4) was strikingly different from that of the starting material. The recovery of LDL in fraction I was greatly reduced and a new broad band was observed with major peaks at pZ 4.9 and 5.15. The peak with a pZ of 4.6 (Fig. 1) was especially reduced in magnitude following Pronase treatment and was apparently converted to less negatively charged products. This peak may contain protein moieties rich in sialic acid since earlier enzymatic studies by Margolis and Langdon (14) showed that the sialic acid-containing regions of the protein are preferentially removed from LDL on treatment with Pronase. The results also suggest that the IEF technique may be useful for separation and isolation of the different proteolysis-resistant products of LDL digestion for further characterization. Finally, despite earlier contradictory reports which question the suitability of preparative isoelectric focusing technique for the study of lower density lipoproteins, it appears that this technique can be applied effectively to the separation of VLDL and LDL. Precipitation of lipoprotein can be avoided by selection of proper experimental conditions. The results also suggest that VLDL and LDL contain isoelectrically heterogeneous subspecies, which are not artifacts of Ampholine binding, findings that further support the concept of discrete lipoprotein heterogeneity (15). ACKNOWLEDGMENTS This work was supported by Grant No. HL 15930 from the National Institutes of Health, United States Public Health Service. The technical assistance of Ronald Jones is gratefully acknowledged.
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REFERENCES 1. Blaton, V., and Peeters, H. (1969) Protides Biol. Fluids, 707-713. 2. Peeters, H., and Blaton, V. (1970)in Atherosclerosis (Jones, R. J., ed.), p. 182, SpringerVerlag, New York. 3. Albers, J. J., and Scanu, A. M. (1971) Biochim. Acta 236, 29-37. 4. Scanu, A. M.. Edelstein, C., and Aggerbeck, L. (1972) Ann. N. Y. Acad. Sci. 209, 311-327. 5. Pearlstein, E., and Aladjem, F. (1972) Biochemistry 11, 2553-2558. 6. Sundaram, G. S., Sodhi, H. S., and Mackenzie, S. L. (1972) Proc. Sot. Exp. Biol. Med. 141, 842-845. 7. MacKenzie, S. L., Sundaram, G. S., and Sodhi, H. S. (1973) C’lin. Chim. Acta 43, 223 -229. 8. Sundaram, G. S., MacKenzie, S. L., and Sodhi, H. S. (1974) Biochim. Biophys. Acta 337, 196-203. 9. Sundaram, G. S., MacKenzie, S. L., and Sodhi, H. S. (1975) Biochim. Biophys. Acta 388, 349-352. 10. Instruction Manual, LKB-Produkter AB, Stockholm-Bromma 1, Sweden. 11. Bennett, B., Swift, L., Gray, M., and LeQuire, V. (1975) Circulation 52(Suppl. 2), 142. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (l%S)J. Biol. Chem. 193, 265-275. 13. Baumann, G., and Chrambach, A. (1975) Anal. Biochem. 64, 530-536. 14. Margolis, S., and Langdon, R. G. (1965)J. Biol. Chem. 241, 485-493. 15. Kostner, G., and Alaupovic, P. (1972) Biochemistry 11, 3419-3428.
BIOCHEMISTRY
88,
425-433 (1978)
Preparative Isoelectric Focusing of Human Serum Low-Density and Low-Density Lipoproteins K.M.
SHANMUGAG.SUNDARAM,
MOHAMED~HAKIR,AND MARGOLIS
SIMEON Departments
of Medicine
Very
and Physiological Medicine, Baltimore,
Chemistry, Maryland
The Johns 21205
Hopkins
School
oj
Received June 1, 1977; accepted March 17, 1978 Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF). IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [W]Ampholine, and gel filtration completely separated the lipoproteins from [W]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.
Several workers have used isoelectric focusing (IEF)’ to study the apoproteins of very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Blaton and Peeters (1) separated human apo-LDL into a major component with a pZ value of 5.36 and a minor one with a pZ value of 6.12, both unreactive with antisera against human LDL. They concluded that apoprotein B consists of two different polypeptides (2). Albers and Scanu (3) used the IEF technique to separate human apo-VLDL into four different polypeptides, each with distinct pZ values and chemical properties. However, when IEF was used to study intact lipoproteins and partially delipidated lipoproteins obtained by extraction with organic solvents, the lipoproteins and apoproteins tended to precipitate at their isoelectric points. These results led Scanu et al. (4) to suggest that IEF has limited value as a preparative technique for intact lipoproteins. On the other hand, Pearlstein and Aladjem (5) successfully applied the preparative technique to the study of intact VLDL in the presence of 33% ethylene glycol. They showed the presence of five distinct subpopulations of particles, one of which was not reactive with anti-LDL antiserum. We have previously shown (6-9) that use of high concentrations of ’ Abbreviations used: VLDL, very low-density lipoproteins; LDL, low-density lipoproteins; HDL, high-density lipoproteins; pl, isoelectric points; IEF, isoelectric focusing. 425
0003-2697/78/0882-0425$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
426
ST WDARAM,
SHAKIR,
AND
MARGOLIS
Ampholine prevents precipitation during preparative IEF studies of intact human serum high-density lipoprotein (HDL) and its subfractions HDLz and HDL3 and that the separated subspecies are not artifacts. When the same technique was applied to the study of intact VLDL and LDL, precipitation and nonreproducible resolution profiles invariably resulted. Neither 33% ethylene glycol nor other stabilizing agents, except glycerol, completely preventrId the precipitation. In this paper, we describe an adaptation of the preparative IEF technique that gives reproducible separation profiles for intact VLDL and LDL. We also present evidence that the observed optical density peaks are not artifacts arising from interactions between the lipoproteins and the Ampholines used for electrofocusing. Finally, we have shown that preparative IEF can be used to isolate different proteolysis-resistant products of LDL digestion. EXPERIMENTAL
PROCEDURE
Materials. [14C]Ampholine, pH 3- 10 (carboxylic groups labeled; specific activity, 5 &i/g), and Ampholine, pH 4-6, were obtained from LKBProdukter AB, S-161 25 Bromma 1, Sweden. Chloramphenicol was obtained from Parke-Davis Co. Triton X-100 and immobilized Pronase were obtained from J. T. Baker Chemical Co. and Sigma Chemical Co., respectively. All other chemicals and solvents were of reagent grade. Preparation of VLDL and LDL. Two- to three-day-old human serum was pooled from two donors to the blood bank of the Johns Hopkins Hospital. After sodium azide (0.05%), chloramphenicol (50 pg/ml), and EDTA (1 mM) wertr added, chylomicrons were removed by centrifugation at 10,000 rpm in a Ti-50 rotor for 30 min in a Beckman Model L2-50 preparative ultracentrifuge. Subsequently, the chylomicron-free serum was centrifuged at 40,000 rpm for 18 hr in a Ti-50 rotor, and the VLDL fraction was removed by tube slicing. VLDL was purified by two subsequent ultracentrifugal washings under similar conditions with a large excess of 0.85% NaCl-0.05% EDTA solution (PH 7.4). The VLDL-free serum was raised to a density of 1.019 g/ml with solid NaBr, and the intermediate density lipoproteins were removed by centrifugation as described above, The infranatant was centrifuged once again under similar conditions to remove completely any contaminating intermediate density lipoproteins. The density of the infranatant was then raised to 1.05 g/ml with NaBr, and the LDL in the density range 1.019- 1.05 g/ml was floated by centrifugation at 45,000 rpm for 24 hr. LDL was purified by two subsequent ultracentrifugal flotations using salt solutions of density 1.05 g/ml. The purified LDL was dialyzed against 0.85% NaCl-0.05% EDTA solution (pH 7.4) at 4°C and stored in the dark in sealed vials under N2 at 4°C until use. Isoelectricfocusing. Isoelectric focusing was performed at 4°C using 2%
ISOELECTRIC
FOCUSING
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421
carrier ampholytes (pH 4-6) and an LKB Model 8101 electrofocusing column as described previously (7). Initially, solutions and density gradients were prepared as described by LKB Instrument Co. (10) without the addition of any stabilizing agent. In later experiments, distilled water used for the preparation of density gradient solutions was replaced by solutions containing ethylene glycol (10, 20, 33, or 40%), Triton X-100 (O.OS-0.5%) (ll), Triton X-100 (0.05%) and glycerol (5%), or glycerol (10%). Applied samples of VLDL or LDL also contained the same amount of stabilizing agent. In experiments using [‘*C]Ampholine, a final concentration of 2% Ampholine was prepared by the addition of 4 ml of unlabeled Ampholine (pH 4-6) to 1 ml of [14C]Ampholine (pH 3- 10). VLDL or LDL, containing 1 mg of protein as determined by the method of Lowry ST al. (12), was applied to the column. The initial and final voltages and currents were 400 V and 2.5 mA and 800 V and 2 mA, respectively, for both VLDL and LDL. Equilibrium was usually reached hi tween 48 and 60 hr, by which time the pH gradient and protein positions had stabilized. The initial and final voltages varied with the stabilizing agent present, but a constant power of 1.5 to 2.5 W was applied. After each run, approximately 80 fractions (1.5 ml) were collected. The pH and absorbance at 280 nm of the fractions were determined at room temperature and in the presence of sucrose using a Fisher Model 325 pH meter and a Beckman Model DU-2 spectrophotometer. Measurements were made immediately after the fractions were collected to minimize intera,.:Lion of atmospheric COz with the fractions. Gel$filtrution. After exposure to [14C]Ampholine for 24 hr at 4”C, VLDL or LDL was chromatographed on Sephadex G-50 or LKB Ultragel AcA 34 columns (1.6 x 40 cm); 2-ml fractions were collected. Optical density of each fraction was determined as described above. Radioactivity in aliquots of these fractions and in fractions from electrofocusing experiments in the presence of [‘4C]Ampholine was determined using a Beckman Model LS 3150 P scintillation spectrometer. Proteolysis of LDL. LDL in phosphate buffer (1.1 M, pH 7.6) was digested with 1% immobilized Pronase at 37°C with gentle shaking for 20 hr. After the Pronase was removed by two centrifugations at 3000 rpm for 30 min, the clear supernatant was dialyzed overnight against three changes of a solution of 0.85% NaCl-0.05% EDTA (pH 7.4) and subjected to preparative IEF in the presence of 10% glycerol as described above. RESULTS
AND DISCUSSION
In the absence of any stabilizing agent, preparative IEF of intact VLDL or LDL resulted in the formation of one or mo;. llocculant rings in the column. The degree of flocculation increased in proportion to the amount of sample applied. The flocculation or “precipitation” was re-
428
SUNDARAM,
SHAKIR,
AND
MARGOLIS
versible. The “precipitate” eluted from the column as an opaque solution that could not be made optically clear by filtration or centrifugation at 2000 rpm for 2 hr. When diluted with an equal volume of 0.05 M carbonatebicarbonate buffer (pH 9.6), the solution became optically clear. The formation of one or more opaque rings was attributed to the accumulation of aggregated VLDL or LDL particles at their isoelectric points. It was possible to minimize this “precipitation” and to obtain fairly reproducible optical density profiles of the eluted fractions by using 0.5 mg or less of VLDL or LDL protein. Methods that lead to precipitation are undesirable since some of the “precipitate” may stick to the column, sediment or float, and elute with later fractions. As a consequence, the optical density profile of the lipoprotein will vary from one experiment to another. The irreproducible results obtained in our early experiments with VLDL and LDL (data not shown) prompted an investigation of stabilizing agents to prevent “precipitation.” The lirst agent tried was 33% ethylene glycol which Pearlstein and Aladjem (5) used for the IEF of VLDL. They dialyzed native VLDL exhaustively against 33% ethylene glycol and added it to the electrofocusing column in a linear sucrose gradient containing 33% ethylene glycol. In our experiments dialysis of either VLDL or LDL against 33% ethylene glycol usually resulted in the deposition of a fine precipitate in the dialysis bag. Our IEF experiments were carried out as outlined by Pearlstein and Aladjem (5), but with a total of 2 to 3 W of power, since it was felt that the 7.5 W apparently used by these authors may denature lipoproteins. Irrespective of whether VLDL dialyzed against 33% ethylene glycol or the clear supernatant obtained by centrifugation of VLDL dialyzed against 33% ethylene glycol was used, “precipitation” was observed within 24 hr. Pearlstein and Aladjem (5) do not mention the appearance of any “precipitate” during IEF of VLDL. The elution profile they reported for native VLDL also suggests that some precipitation might have occurred since most of the lipoprotein eluted in a single peak, comprising tube numbers 60 to 140, with an optical density greater than 2.0. Different concentrations of ethylene glycol (10, 20, or 40%) were not completely effective in preventing the “precipitation.” Triton X-100 (11) (O.OS-0.5%) was equally ineffective. Although the use of Triton X-100 (0.05%) and glycerol (5%) appeared to prevent the precipitation, a faint opaque ring was noted and isoelectric profiles were not reproducible. Glycerol (10%) completely eliminated the “precipitation” (delined as the appearance of a visible flocculant ring) and gave optical density profiles for LDL and VLDL, as shown in Figs. 1 and 2, respectively. In the presence of 10% glycerol, as much as 6 mg of LDL or VLDL protein was applied to the column without any precipitation. Glycerol at 5 and 2.5%
ISOELECTRIC
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1 6
$0.2
I
2
-51 a
0
10
20
30
40 50 60 Tube Number
-2000
-3
-coo
-J
80
70
-4
100
1 -c ‘E B IJ
FIG. 1. Isoelectric focusing profile of low-density lipoprotein in the presence of 10% (w/v) glycerol. (~1 Azso nm; (0) “C activity; (0) pH. The isoelectric protile is typical or those obtained from three different experiments carried out at 4-day intervals using the same preparation of LDL. The isoelectric focusing time was 60 hr. Fractions containing about 1.6 ml (30 drops) were collected.
did not completely prevent precipitation with the 6-mg protein load. It is not known whether 10% or greater concentrations of glycerol will prevent lipoprotein precipitation at higher sample loads. The optical density profiles shown in Figs. 1 and 2 were highly reproducible in three different consecutive experiments (Table 1) when electrofocusing was carried out for 60 hr. Electrofocusing for a longer period (84 and 96 hr) did not alter the optical density profiles or pH proflies, showing that the pH positions occupied by the proteins were true p1
-7 6 I 51 a -4 -3 0
10
20
30
c--rcI 40 50 60 Tube Number
70
c,
80
\. 90
( 100
I 5 1000fj u^
2030
_
FIG. 2. Isoelectric focusing protile of very low-density lipoprotein in the presence of 10% (w/v) glycerol. (x)A,@ “,,,; (0) “C activity; (0) pH. The isoelectric profile is typical of those obtained from two different experiments using the same preparation of VLDL. Fractions containing about 1.3 ml (25 drops) were collected.
430
SUNDARAM,
SHAKIR, TABLE
ISOELECTRIC
Experiment
POINTSOF
AND MARGOLIS 1
LDL AND VLDL FRACTIONS"
Fraction I
Fraction II
LDL A B C
4.10 4.15 4.15
4.50 4.50 4.50
4.60 4.60 4.60
4.98 5.00 4.98
5.22 5.22 5.22
5.30 5.30 5.30
5.35 5.35 5.35
5.45 5.45 5.45
6.10 6.05 6.10
VLDL A B
4.20 4.20
4.40 4.45
4.55 4.55
4.90 4.96
5.05 5.05
5.15 5.15
5.40 5.40
5.45 5.45
6.10 6.10
a The pZ values represent the pH values of fractions containing the peaks shown in Fig. 1 or 2. Recovery of LDL and VLDL from the column ranged from 96 to 100%. The lipoproteins were distributed in the following proportions: LDL, 36% in Fraction I and 64% in Fraction II; VLDL, 73% in Fraction I and 27% in Fraction II.
values. Furthermore, the current drawn from a constant-voltage power supply was at minimum after 60 hr, showing that the electrofocusing was completed by that time (10). There was no further drop in the current applied to the column on continuing the electrofocusing for 6 more hr, indicating that a true steady-state equilibrium was present. Based on the optical density profile shown in Fig. 1, the eluted material from the IEF of LDL was arbitrarily divided into fractions I and II. Figure 2 clearly shows a similar distinct separation of fraction I from fraction II in VLDL. The smaller peaks of VLDL with pZ values between 4.90 and 6.10 were included in fraction II because of their resemblance to peaks in fraction II of LDL. It is apparent from comparing the isoelectric profiles of LDL and VLDL that several peaks with similar pZ values appear in both lipoproteins. The isoelectric profiles of peaks with pZ values 4.10,4.60, and 6.10 in LDL were strikingly similar to those of peaks with pZ values 4.20, 4.55, and 6.10 in VLDL. The magnitude of most peaks in LDL is greater than the magnitude of peaks with similar pZ values in VLDL. Several prominent peaks in LDL have no counterpart (for example, pZ 5.33) or very small peaks (pZ 5.4) in VLDL. Both LDL and VLDL have some peaks that are not fully resolved from one another. Further electrofocusing experiments using a narrower-pH range Ampholine may allow complete resolution of these peaks. Table 1 shows the excellent reproducibility of the pZ values in different experiments using the same preparation of LDL or VLDL. Neither the resolution pattern nor the pH values of the peaks varied when the experiments were carried out under identical conditions at 4-day intervals. Even though several peaks with different pZ values were observed in the iso-
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electric profiles of HDL, HDL*, and HDL3 (6-9), the IEF profiles of highdensity lipoproteins are quite different from those of LDL and VLDL. Considering the heterogeneity of VLDL particles, the presence of several isoelectrically different species is not surprising. However, the apparent isoelectric heterogeneity of LDL was unexpected since LDL is generally considered to consist of rather homogenous particles. The isoelectric heterogeneity of LDL and VLDL suggest that, when these lipoproteins are isolated over a particular density range by ultracentrifugation, they may consist of several subspecies which differ in specific apoprotein content, lipid/protein ratios, or sialic acid or other carbohydrate components. Purification and characterization of each peak is necessary to understand the significance of the observed heterogeneity. However, any hypothesis about the structure of VLDL and LDL must take into account and explain the heterogeneous behavior of these particles on IEF. Many sources of artifact in IEF have been considered (6-9,13). Of greatest concern, however, are possible interactions between Ampholine and the lipoproteins. Therefore, we added labeled Ampholine to lowdensity lipoproteins in experiments similar to our earlier work with highdensity lipoproteins (9). Figures 1 and 2 show the results of electrofocusing of LDL and VLDL in the presence of [14C]Ampholine. Since no 14C activity was eluted with the peaks containing the lipoproteins, it is clear that Ampholine did not bind to either lipoprotein under these conditions of electrofocusing During gel filtration on Sephadex G-50 A.
.60 -
- 6,000 E -5,000 - 4,000
Tube
FIG. 3. Chromatography 2% (w/v) [Y]AmphoIine,
‘E 4 2
Number
on Sephadex G-50 of (A) LDL and (B) VLDL pH 3- 10. (0) A,,, “,,,; (0) 14C activity.
solutions
containing
432
SUNDARAM,
SHAKIR,
AND MARGOLIS
40
Tube
50
60
70
Number
FIG. 4. Isoelectric focusing profile of Pronase-digested LDL in the presence of 10% (w/v) glycerol. (0) AZsO“,,,. Fractions containing about 1.6 ml (30 drops) were collected.
columns both VLDL and LDL eluted with the void volume, whereas all of the labeled Ampholine eluted in a late peak (Fig. 3). We also employed LKB Ultragel AcA 34, a mixture of agarose and polyacrylamide, to examine lipoprotein-Ampholine interactions during gel filtration on a gel other than Sephadex. No significant 14C activity was eluted with the lipoprotein peaks (data not shown). The failure of Ampholine to bind to the lipoproteins in any of these experiments strongly suggests that the heterogeneity observed during electrofocusing is not due to lipoprotein-Ampholine interactions. When LDL was digested with insoluble Pronase and then subjected to IEF, the observed profile (Fig. 4) was strikingly different from that of the starting material. The recovery of LDL in fraction I was greatly reduced and a new broad band was observed with major peaks at pZ 4.9 and 5.15. The peak with a pZ of 4.6 (Fig. 1) was especially reduced in magnitude following Pronase treatment and was apparently converted to less negatively charged products. This peak may contain protein moieties rich in sialic acid since earlier enzymatic studies by Margolis and Langdon (14) showed that the sialic acid-containing regions of the protein are preferentially removed from LDL on treatment with Pronase. The results also suggest that the IEF technique may be useful for separation and isolation of the different proteolysis-resistant products of LDL digestion for further characterization. Finally, despite earlier contradictory reports which question the suitability of preparative isoelectric focusing technique for the study of lower density lipoproteins, it appears that this technique can be applied effectively to the separation of VLDL and LDL. Precipitation of lipoprotein can be avoided by selection of proper experimental conditions. The results also suggest that VLDL and LDL contain isoelectrically heterogeneous subspecies, which are not artifacts of Ampholine binding, findings that further support the concept of discrete lipoprotein heterogeneity (15). ACKNOWLEDGMENTS This work was supported by Grant No. HL 15930 from the National Institutes of Health, United States Public Health Service. The technical assistance of Ronald Jones is gratefully acknowledged.
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REFERENCES 1. Blaton, V., and Peeters, H. (1969) Protides Biol. Fluids, 707-713. 2. Peeters, H., and Blaton, V. (1970)in Atherosclerosis (Jones, R. J., ed.), p. 182, SpringerVerlag, New York. 3. Albers, J. J., and Scanu, A. M. (1971) Biochim. Acta 236, 29-37. 4. Scanu, A. M.. Edelstein, C., and Aggerbeck, L. (1972) Ann. N. Y. Acad. Sci. 209, 311-327. 5. Pearlstein, E., and Aladjem, F. (1972) Biochemistry 11, 2553-2558. 6. Sundaram, G. S., Sodhi, H. S., and Mackenzie, S. L. (1972) Proc. Sot. Exp. Biol. Med. 141, 842-845. 7. MacKenzie, S. L., Sundaram, G. S., and Sodhi, H. S. (1973) C’lin. Chim. Acta 43, 223 -229. 8. Sundaram, G. S., MacKenzie, S. L., and Sodhi, H. S. (1974) Biochim. Biophys. Acta 337, 196-203. 9. Sundaram, G. S., MacKenzie, S. L., and Sodhi, H. S. (1975) Biochim. Biophys. Acta 388, 349-352. 10. Instruction Manual, LKB-Produkter AB, Stockholm-Bromma 1, Sweden. 11. Bennett, B., Swift, L., Gray, M., and LeQuire, V. (1975) Circulation 52(Suppl. 2), 142. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (l%S)J. Biol. Chem. 193, 265-275. 13. Baumann, G., and Chrambach, A. (1975) Anal. Biochem. 64, 530-536. 14. Margolis, S., and Langdon, R. G. (1965)J. Biol. Chem. 241, 485-493. 15. Kostner, G., and Alaupovic, P. (1972) Biochemistry 11, 3419-3428.