Preparation and Properties of an Apoprotein Derivative of Human Serum ~ -Lipoprotein 1

A. M. GOTTO, R. 1. LEVY and D. S. FREDRICKSON, Laboratory of Molecular Diseases, National Heart institute, National Institutes of Health, Bethesda, Maryland 20014

ABSTRACT

The aim of this study was to develop a convenient method for the preparation and study of a soluble delipidated form of human serum fl-lipoprotein. This was achieved by succinylation and delipidation with ether-ethanol (3 : 1 ). The succinylated apoprotein was soluble in either 0.13 M Tris-HC1 buffer, pH 8.2 (for fl-lipoprotein prepared by ultracentrifugation) or in the same Tris buffer to which 5 mM sodium decyl sulfate was added (for heparin-Mn precipitated fi-lipoprotein). The immuno- logical activity of fi-lipoprotein or its apo- protein were markedly altered by succiny- lation. Whereas the succinylated fl-lipo- protein appeared as one peak in the analytical ultracentrifuge, the succinylated apoprotein appeared as two. Under the electron microscope fl-lipoprotein and suc- cinylated fl-lipoprotein were indistinguish- able, appearing as uniform preparations of spherical particles 215 to 220 A in diam- e ter.

INTRODUCTION

A LTHOUGH THE LIPID-FREE apoprotein of human serum a-lipoprotein has been ex-

tensively studied, the protein moiety of fl-lipo- protein (fl-LP) has not. Attempts to remove completely the neutral lipids or to strip away phospholipids from fl-LP have usually pro- duced gel-like products which were irreversibly aggregated (1-4). This difficulty in obtaining lipid-free fl-apoprotein has greatly hampered its characterization.

Recently, delipidation in the presence of the detergent sodium dodecyl sulfate (5), reduc- tion and alkylation after delipidation (6) or succinylation (7) prior to delipidation have been reported to yield a more soluble fl-apo- protein. However, the insolubility of sodium dodecyl sulfate interferes with further study of the apoprotein preparation and this detergent cannot be removed completely, even by dialysis for several days (5).

It is the purpose of this communication to

aPresented at the AOCS Meeting, Washington, D. C., April, 1968.

report methods which can conveniently be used for the preparation in high yield of a soluble succinylated delipidated derivative (s-fl-apopro- tein) of human serum fl-LP. Two different methods for preparing large amounts of pure /3-LP are described and compared as to con- venience and properties of final product. The s-~-apoprotein derived from these-two proced- ures is studied and contrasted with respect to aqueous solubility, immunological activity and sedimentation behavior. A portion of this work hasbeen described in preliminary form (8, 9). A report on the properties of s-/3-apoprotein has recently appeared from another laboratory (10) during the preparation of this paper.

MATERIALS AND METHODS

Succinic anhydride was obtained from East- man Kodak Distillation Products Industries, Rochester, N. Y. All materials used were of the highest purity commercially available, ex- cept for sodium decyl sulfate and sodium do- decyl sulfate, which were recrystallized.

All of the lipoprotein and apoprotein frac- tions were extracted in chloroform-methanOl (2:1, v /v ) , for estimation of triglyceride (11 ), cholesterol (12) and phospholipid (13). The lipids in the C / M extracts also were examined by thin-layer chromatography using both polar and nonpolar solvents (14). Protein was de- termined by the method of Lowry et al. (15). Paper electrophoretic (16) and immunological techniques used for the analysis of lipoproteins have been described previously (17, 18). For electrophoresis of s-fl-apoprotein, 1% albumin was omitted from the buffer and 0.01% brom- phenol blue was used as the protein stain. Anti- bodies to s-fl-LP, or s-/?-apoprotein were pro- duced by three serial injections into the foot pads of white New Zealand rabbits of 1.25 ml of the antigen (5 to 8 mg protein) mixed with 0.75 ml of Freund's adjuvant (17). The anti- sera to s-fl-LP, designated SB, and SB v and to s-fl-apoprotein (SDB~ and SDB~) were har- vested as previously described (17, 18). They reacted weakly and inconstantly with native fl-LP. All four antisera strongly reacted with s-fl-LP and SB1, SDB1, and SDB~ reacted with s-fi-apoprotein. The antibodies to B-LP which were used in this study have been characterized

463

464 A . M . GOTTO, R. 1. LEVY AND D. S. FREDRICKSON

300

c~ ~ 200

J

~EOO

2 0 0 400 600 800 I000 1200 1400 1500 t600 SUCCINIC ANHYDRIDE ADDED (~moles)

FIG. 1. Succinylation of fl-lipoprotein. The dot- ted line represents the total number of amino groups present before addition of succinic anhy- dride. The differences between this value and the number of amino groups determined in subsequent samples were used to measure the quantity of amino groups succinylated at a particular time. After the addition of a measured quantity of suc- cinic anhydride, 2 N NaOH was added with an automatic titrator until the pH was constant. An aliquot was then obtained for assay of free amino groups.

and descr ibed previous ly ( 1 7 ) . All an t i sera were tested against a b l a n k con ta in ing sod ium

3

--2 z

g~

<3

I B ) 2 5 4

DETERGENT CONCENTRATION (raM)

FIG. 2. Effects of sodium decyl sulfate and sodium dodecyl sulfate on the aqueous solubility of s-fl-apoprotein prepared from heparin and man- ganese precipitated fl-LP. Three mg of s-fl-LP were delipidated (8) . To the dry residue was added 1 ml of 0.13 M Tris buffer, pH 8.2, which contained varied quantities of sodium decyl sul- fate (C)) or sodium dodecyl sulfate ( e ) . Non- succinylated fl-apoprotein was incubated with Tris buffer containing 2 mM sodium decyI sulfate ( & ) . The samples were incubated for 4 hr at 37C and aliquots of the aqueous phase were removed for protein assay. All of the visible residue from the succinylated preparat ions was soluble at concen- trations of detergent of 2 mM or greater.

TABLE I Solubility of Succinylated Apoprotein from /%Lipoprotein

Prepared by Ultracentrifugation: Effect of Succinylation a

Incubation time (hr) of Soluble proteinb

s-apoprotein Fr action (rag)

4 Succinylated B- 3.9 ~+ 0.1 lipoprotein

16 Non-succinylated 0.8 • 0.03 B-lipoprotein

a Succinylation, delipidation and solubilization of the de- lipidated products were performed as previously described (8).

bThese values represent the total protein solubilized after delipidation of 24 individual samples -1- s t a n d a r d error. The quantity of suceinylated or native B-lipoprotein delipidated in each flask was 4.3 me. Incubation was at 37C.

decyl sulfate w h e n this subs tance was present , s ince it was no t ed t h a t several an t i sera fo rmed h a z y prec ip i t in b a n d s wi th this detergent .

Nega t ive ly s ta ined p repa ra t ions of f l -LP or s-/?-LP were ob ta ined on a F o r m v a r - e a r b o n coa ted grid us ing 2 % po tass ium p h o s p h o t u n g - state, p H 7.0. T h e l ipopro te in solut ions con- t a ined 0.1 m g of p ro t e in per ml.

F o r the d e t e r m i n a t i o n of s ed imen ta t ion c o - efficients, d is tances f r o m the m a x i m u m ord ina te to the r e fe rence hole (x) were m e a s u r e d wi th a N i k o n m i c r o c o m p a r a t o r . T h e sed imen ta t ion coefficient (s) was ca lcula ted f r o m the slope of the l ine w h e n log x was plot ted against time. Values of s were cor rec ted to s t anda rd condi- t ions ( 1 9 ) , a s suming a v of 0.971 for s-f l -LP (20 ) and 0.727 for s - f l -apoprote in . The fo rmer va lue was ob ta ined f r o m the rec iproca l of the average h y d r a t e d dens i ty while the la t te r value was ca lcula ted f r o m the amino acid composi- t ion (5) by the m e t h o d of S c h a c h m a n (21) and neglected b o u n d sod ium decyl sulfate. A1-

TABLE 11 Solubility of Succinylated Apoprotein from Heparin and

Manganese Precipitated fi-Lipoprotein: Effect of Sodium Decyl Sulfate a

Total incubation time (hr) of s-apoprotein Soluble protein

4.5 0.4 + 0.02 16 Gel b

Sodium decyl sulfate added (5 mM) e

20 3,8 • 0.1

aFor details, please see explanatory notes for Table I. The quantity of succinylated fl-LP delipidated in each flask was 4.4 me. The total number of individual samples ex- tracted was 24.

bAccurate pipetting from the gel was not possible. carter 16 hr, sodium decyl sulfate was added to a final

concentration of 5 raM. Incubation at 37C was continued for another 4 hr~

LIPmS, V o L 3, No. 6

SUCCINYLATED fl-APOPROTEIN 4 6 5

Fro. 3. Heterogeneity of s-/3-LP on immunoelectrophoresis in 0.5% agarose. Samples of s-fi-LP were placed in the 4 center wells and subjected to electrophoresis for 40 min. The following antisera were used: left trough A, R,; right trough A, SDB1; left trough B, R~; right trough B, Rs; left trough C, SB~; right trough C, SB~; left trough D, RT; right trough D, S1A (17).

though these values of v are those of the non- suCcinylated lipoprotein and apoprotein, the recent data of Scanu et al. (10) would seem to justify their use in calculations involving the succinylated derivatives. A plot of 1/$20,~ vs. concentration was used for extrapolation to zero concentration, employing seven concen- trations of protein (as s-fl-LP or s-fl-apopro- tein) over a range from 1 to 5 mg. Approxi- mate values were obtained for K from the expression, S20 ' w = S~ w (1-Kc) . These values were 48 and 111 ml /gm for ultracentri- fugally prepared s-fi-apoprotein in the presence and absence of 20 m M sodium decyl sulfate, respectivelyl and 9.2 ml /gm for s-f l-LP.

The solutions of NaC1, NaBr and Tris-HCl buffer which were used in the preparat ion of fl- LP, s-fl-LP and s-fl-apoprotein c o n t a i n e d 0.01% EDTA, adjusted to pH 7.0. The final pH values of the 0.15 M NaC1-EDTA and of

the 0.13 M Tr is -EDTA solutions were adjusted to 7.0 and 8.2, respectively.

Plasma was collected from fasting, normal subjects by plasmaphoresis and stored at 4C.

TABLE 1II Composition of fi-lipoprotein and Succinylated

p-apoprotein a

% Composition Phospho- Triglyc- Choles-

Preparation Protein lipid eride terol

/3-LP by ultra- eentrifugation 24:2 25.'1 4.7 46.4

B-I:.P by heparin -Mn precipitation 23.7 22.2 5.0 49.1

s-fi-apoprotein no. 1 98.4 1.5 0.0 <0.1

s-fl-apoprotein no. 2 98.7 1.2 0.0 <0.1

s-B-apoprotein �9 no. 3 98.8 1.l 0.0 <0. I

aAll analyses were done fn dupli6ate.

LiPiDS, VOL. 3, i ~ . 6

466 A . M . GOTTO, R. I. LEVY AND D. S. FREDRICKSON

Fractionation was usually begun within two weeks of collection. Procedures for the prep- aration of fl-LP by precipitation with heparin and manganese in conjunction with ultracen- trifugal separation between densities 1.019 and 1.063 (22), for succinylation and for delipida- tion have been described (8). fl-LP was extensively succinylated with relatively small quantities of succini c anhydride (Fig. 1 ). Simi- lar methods were employed when fl-LP was prepared by ultracentrifugation alone, except that treatment with thrombin and precipitation with heparin and manganese were omitted. All fl-LP used for this study was immUnochemical- |y pure .

The dry residue of s-/?-apoprotein, prepared as previously described (8), was incubated at 37C with 0.13 M Tris-HC1 buffer, pH 8.2 or with the same Tris buffer containing 5 mM sodium decyl sulfate. If higher concentrations of fl-LP were delipidated, or rarely with low concentrations prepared with heparin and man- ganese, it was necessary to increase the con-

centration of sodium decyl sulfate to 20 mM to obtain complete solubilization.

RESULTS

Solubility of S-fl-Apoprotein Succinylation of fl-lipoprotein led to a mark-

ed increase in solubility after delipidation with ether-ethanol (Table I ) . The method of prep- aration significantly affected the solubility of the s-fl-apoprotein. When fl-LP was prepared solely by ultracentrifugation, rapid and com- plete solubilization of the s-/?-apoprotein was achieved. All of the protein recovered after delipidation was solubilized by this procedure. On the other hand, when precipitation with manganese and heparin was employed, only partial solubilization was achieved and gel for- mation occurred (Table I I ) .

Complete solubilization of the s-fl-apoprotein obtained by heparin and manganese precipita- tion was made possible by the further addition of 5 mM sodium decyl sulfate (Table I I ) . Sodium decyl sulfate and sodium dodecyl sul-

FIG. 4. Immunodiffusion patterns of fl-LP and s-fl-LP. The following antisera were placed in center wells: SB~ in A, SB: in B, SDB1 in C, SDB~ in D, R~ in E, Hyland anti-/? in F, SIA in G and ~-LpT in H (17). The following antigen preparations were placed in the outer wells: heparin and manganese precipitated /?-LP in 1 and 4, ultracentrifugally prepared fl-LP in 2 and 3, s-/?-LP from precipitated lipoprotein in 5 and 8, and s-/?-LP from ultracentrifugally prepared lipoprotein in 6 and 7~

LIPIDS, VOL. 3, NO. 6

SUCCINYLATED p-APOPROTEIN 467

Fl~. 5. Immunodiffusion patterns of p-LP. Effects of succinylation and delipidation. Antiserum SB1 is in the central well. In outer wells clockwise from 1 are P-LP (1, 2), s-/?-apoprotein (3-6) and s-/?-LP (7, 8).

fate were equally effective in promoting solu- bilization (Fig. 2). Under these experimental conditions, there was little solubility of the delipidated material in the presence of 2 X 10 -3 M sodium decyl sulfate unless the lipo- protein was succinylated. I t was not necessary for the detergent to be present, however, dur- ing the delipidation procedure in order to achieve solubilization.

Composition of the Beta-LP and s-/~-Apoprotein

No significant differences were found between the preparations of p-LP which were precipi- tated with heparin and those which were not. The extraction procedure removed more than 98 % of the total lipid content o f /? -LP (Table I I I ) . No cholesterol (free or esterified) or triglyceride was detected by thin-layer chro- matography. A small quantity of phospholipid, usually from 1.1% to 1.5%, could be detected.

Electrophoretic and immunological Properties of Beta-LP, S-p-LP and S-p-Apoprotein

Succinylation of /?-LP from either method of preparation resulted in a more rapid electro- phoretic migration on both albumin impreg- nated paper or on 0.5% agarose. On paper electrophoresis s-/?-apoprotein migrated more slowly than s-fl-LP and had a mobility similar to albumin.

Some, but not all, of the rabbit and sheep antisera to native /?-LP retained activity with s-/?-LP when examined by immunoelectro- phoresis on 0.5% agarose. Heterogeneity of s-/?-LP was indicated by the presence of at least two precipitin arcs of different mobility with antisera SB1, SB~ and SDB1 (Fig. 3). Some antisera to /?-LP reacted with s-/g-LP, e.g., R4, R 7 and S~A, while others did not, e.g., R 2 and R 8 (Fig. 3). Those antisera to p-LP which did react, however, formed only the more slowly migrating precipitin arc.

LIPIDS, VOL. 3, NO. 6

468 A . M . GOTIO, R. I. LEVY AND D. S. FREDRICKSON

FiG. 6. Sedimentation velocity pattern of s-fi- LP. The lipoprotein was prepared with heparin and manganese precipitation. The protein concen- tration was 0.9 mg/ml, the solvent 0.13 M Tris buffer, pH 8.2, and the bar angle 75 ~ The photo- graph was taken at 90 min after reaching speed. Temperature was 20C.

Beta-LP prepared by either heparin and manganese precipitation and ultracentrifuga- tion or by ultracentrifugation alone was tested against a large number of antisera by the double-diffusion t e c h n i q u e of Ouchterlony. Complete immunological identity of the lipo- protein preparations derived by these two meth- ods was indicated (Fig. 4). Likewise, the succinylated derivatives of fl-LP from these two types of preparations exhibited immunological identity (Fig. 4). The succinylated derivative, however, showed only partial immunological identity with the parent fi-LP. As with im- munoelectrophoresis, s-fi-apoprotein r e a c t e d with certain antisera prepared against itself or against s-fl-LP, and reacted inconstantly with a few, but not most antisera to fl-LP. The immunological heterogeneity of s-fl-LP again was demonstrated by the appearance of two precipitin lines with the antiserum SB1, one of

LIPIDS, VOL. 3, NO. 6

which was immunologically identical with the precipitin line of s-/?-apoprotein (Fig 5).

Sedimentation Studies

Both fl-LP and s-fi-LP (Fig. 6) sedimented as single components in the analytical ultra- centrifuge in 0.13 M Tris-HC1 buffer (0.01% EDTA) , pH 8.2. The So20 ' ~ value of the latter was 5 to 6. In sedimentation velocity experi- ments s-/?-apoprotein exhibited heterogeneity independently of the method of preparation of fl-LP and the presence or absence of sodium decyl sulfate (Fig. 7). The So20 ' ,~ values of s- /?-apoprotein from heparin-Mn precipitated fl- LP were 4.2 and 5.6 for the slow and fast peaks of the preparation shown in Fig. 7 (in 20 mM sodium decyl sulfate). Although there were variations in the sedimentation coefficients with different preparations, two or more components were present in all instances. The So20 ' ,~ values of ultracentrifugally prepared s-fi-apoprotein were 3.3 and 6.0 for the slow components in the presence and absence of 20 mM sodium decyl sulfate, respectively. Although a fast component was definitely present, it was not possible to measure accurately its So20 ' ~ value. Sedimentation in 0.13 M Tris-HC1 buffer (pH 8.2), which contained 0.15 M NaCI gave quali- tatively similar results to those described above.

Electron Microscope Studies

Electron micrographs, prepared with the negative staining technique, indicated no differ- ences between fi-LP prepared by ultracentri- fugation alone or prepared by an initial preciPi- tation step. A typical preparation uniformly contained spherical particles of 215 to 220 A in diameter (Fig. 8). Succinylation did not alter the appearance of the lipoprotein particles.

DISCUSSION

The observation of Margolis and Langdon that diazotization of fl-LP increases the solu- bility of the delipidated product (3) suggested to us that the addition of negative charge might increase the solubility of fi-apoprotein. The results with succinylation provide strong sup- port for this concept. When succinylation itself is insufficient to achieve solubilization of the apoprotein, viz. for fi-LP obtained by heparin and manganese precipitation, the further addi- tion of 5 mM sodium decyl sulfate permits complete solubilization. In comparison with sodium dodecyl sulfate, the decyl sulfate has the advantages of greater aqueous solubility and can be completely removed by dialysis. The mechanism of the decrease in aqueous solubility of the succinylated apoprotein from

SUCCINYLATED fi-APoPROTEIN 469

FIG. 7. Sedimentation velocity patterns of s-fi-apoprotein. Experimental conditions were as fol- lows: left frame, s-fl-apoprotein from heparin-Mn treated fl-LP, 4.4 mg protein per ml in 0.13 M Tris HC1 buffer, 20 mM sodium decyl sulfate (pH 8.2), time after reaching speed was 130 min; mid- dle frame, s-fl-apoprotein from ultracentrifugally prepared B-LP, 4.0 mg protein per ml same solvent as above, time after reaching speed was 161 min; right frame, s-fl-apoprotein from ultracentrifugally prepared fl-LP, 4.2 mg/ml in 0.13 M Tris HC1 buffer (oH 8.2), time after reaching speed was 129 rain. For all three experiments, the bar angle was 75 ~ and the temperature 20C.

heparin and manganese precipitation is not understood (Table I ) .

Although precipitation with heparin and manganese alters the solubility of delipidated fl-LP, no alterations are detected in the im- munological properties of the fl-LP, and its succinylated and delipidated derivatives. Suc- cinylation, which may introduce a strong hap- tenic group, alters the antigenic character of fi-LP such that it reacts weakly with antisera to fl-LP and shows only partial immunological identity with the parent molecule (Fig. 4). These results are in contrast to a recent report by Scanu et al. (10), in which s-fl-LP failed to react with antisera to fl-LP. It is not sur- prising, therefore, that delipidation of s-fi-LP almost completely abolishes activity with most antisera to fl-LP, although s-fi-apoprotein re- acts strongly with certain antisera to itself or to s-fi-LP.

Heterogeneity of s-fl-apoprotein is clearly demonstrated by sedimentation studies with s-fl-LP. The finding of heterogeneity was a consistent one with all preparations, whether or not heparin and manganese precipitation was employed in the preparative procedure (Fig. 7) . Previous failure to note heterogeneity of s-fi-apoprotein in sedimentation experiments (5) may have been caused by the apparent

homogeneity during the relatively early and middle stages of observation in the ultracen- trifuge. This is particularly important at high concentrations of protein. Although a signifi-

Fro. 8. Electron micregraph of fl-LP with nega- tive staining technique. A solution of heparin and manganese precipitated fl-LP (0.1 mg/ml) was used. The magnification was 300,000.

LIPIDS, VOL. 3, NO. 6

470 A . M . GOTTO, R. I. LEVY AND D. S, FREDRICKSON

cant decrease in the value of S~ ' w occurs when sodium decyl sulfate is added, ultracen- trifugal heterogeneity is not dependent upon the presence of this detergent. Heterogeneity of s-fl-apoprotein, recently described in sedi- mentation equilibrium and gel filtration experi- ments, has been attributed to aggregation (10).

Application of the methods described in this communication should facilitate study of a de- rivative of fl-apoprotein by further application of other techniques of protein chemistry.

ACKNOWLEDGMENT

We thank Alan Rosenthal for performing electron mic- roscope studies, Mariel Birnbaumer for expert technical assistance and Senye Temel for assay of phospholipid and thin-layer chromatographic analyses. Sodium decyl sulfate and sodium dodecyl sulfate were gifts of Ralph Reisfeld and the E. I. duPont Co., respectively.

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[Received April 8, 1968]

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