Analysis of lipoprotein apoproteins by SDS-gel filtration column chromatography

Transcription

1 Anlysis of lipoprotein poproteins by SDS-gel filtrtion column chromtogrphy Chrles E. Sprks nd Julin B. Mrsh The Medicl College of Pennsylvni, Deprtment of Physiology nd Biochemistry, Phildelphi, PA Abstrct Rt plsm, contining1251-lbeledtriglyceriderich lipoprotein, ws mixed following lipid extrction with 10% SDS buffer nd nlyzed by gel filtrtion chromtogrphy on columns using n elution buffer contining 1% SDS. Lbeled poproteins were seprted into po B, po E, nd po C rdioctivity peks. Lbeled peptides, tyrosine, nd iodide werelsoresolved by this method. solted lipoprotein frctions were seprted into the sme components. The method offers the dvntges of quntittive rdioctivity recovery, lrge smple volume, nd resolution of two po B proteins.-sprks, C. E., nd J. B. Mrsh. Anlysis of lipoprotein poproteins by SDSgel filtrtion column chromt0grphy.j. Lipid Res : Supplementry key words poprotein B. po B heterogeneity. triglyceride-rich lipoprotein. VLDL. poprotein turnover problems my be encountered. Gels stined with Coomssie blue my show vrible poprotein losses nd inconstnt dye binding of poproteins hmpers clcultion of specific ctivity, nd fctors hve been employed to correct for such differences (23, 24). Apoproteins hve previously been seprted using gel filtrtion column chromtogrphy in the presence of SDS (25-28). The present studies detil the use of SDS columns for seprtion of poproteins of triglyceride rich lipoprotein (TRL) in the presence of plsm protein. mproved resolution llows seprtion of po B into two component proteins. This method hs prticulr ppliction to the study of po B metbolism using injected 1251-lbeled TRL. Lipoprotein turnover studies frequently use lz5lipoprotein lbeling with C1 (1-4), intrvenous injection of the lbeled lipoprotein, nd sequentil plsm smpling followed by ultrcentrifugtion of plsm lipoproteins nd subsequent poprotein nlysis (5, 6). When poprotein-derived peptides occur, trichlorocetic cid solubility mybe used to distinguish intct poproteins from degrded poproteins (7). When po B metbolism is studied, the smple my be extrcted with tetrmethylure in which po B is insoluble (8). Uncertinties, however, exist with such experimentl designs. The use of lz5cl results in vrible lipid lbel (4) nd residul iodide remins ssocited with the lipoprotein even fter extensive dilysis. During ultrcentrifugtion, vrible quntities of individul poproteins my be stripped from the lipoprotein nd enter higher density frctions (9, 10). Lipoprotein delipidtion tny extrct poprotein lbel, especilly po C (11). Tetrmethylure precipittion yields totl po B frction (8, 12) reveling little informtion bout the metbolism of heterogeneous po B subtypes reportedly contined in this frction (13, 14). Polycrylmide gel electrophoresis (PAGE) in the presence of SDS (15, 16), mpholine (17-22), or ure (8) effects good resolution of individul poproteins lthough some MATERALS AND METHODS Preprtion of lipoproteins Lipoproteins were isolted from the ser of mle rts of the Fischer 344 strin weighing g. The rts were fed diet contining 68% (w/w) sucrose, 10% (w/w) vegetble oil, nd 18% (w/w) csein (CN Nutritionl Biochemicls, Clevelnd, OH). The rts were lightly nesthetized by intrperitonel injection of 30 mg/kg nembutl nd blood ws obtined by ortic puncture. Lipoproteins were prepred by ultrcentrifugtion of pooled ser t d < g/ml (23,24). The TRL frctions from which chylomicrons hd been removed were designted VLDL (d < 1.006). n vitro rdioiodinted lipoproteins were prepred using the C1 method s described (1, 2). The lz5lbeled TRL hd less thn one iodine tom per molecule of poprotein nd specific ctivities rnged from 1 to 10 dpm/ng protein. Between 6 nd 35% of the rdioctivity ws in lipid, s estimted by the lipid Abbrevitions: PAGE, polycrylmide gel electrophoresis; SDS, sodium dodecyl sulfte; TRL, triglyceride-rich lipoprotein (d < g/ml contining chylomicrons): VLDL, very low density lipoprotein (d < g/ml); HDL, high density lipoprotein (1.063 < d < g/ml). 514 Journl of Lipid Reserch Volume 22, 1981

2 extrction procedure of Lux, John, nd Brewer (29) s described previously (23, 24). n vivo lbeled lipoproteins were prepred by intrperitonel injection of either 0.5 mci 3H-lbeled L-mino cids (New Englnd Nucler, Boston, MA, NET 250), or intrvenous injection of 0.25 mci of 14C-lbeled L-mino cids (New Englnd Nucler, Boston, MA NEC 445). Lbeled TRL ws isolted 2 hr fter injection of rdioctive mino cids. Preprtion of smples for column nlysis Plsm contining 1251-lbeled lipoproteins ws delipidted using the method of Lux, et l. (29) s described previously (23). Briefly, the method involves dding 1 mg dextrn T-500 (Phrmci Fine Chemicls Co., Pisctwy, NJ) per ml plsm followed by delipidtion. After collection of the precipitte by centrifugtion, the protein pellet ws wshed in pure diethyl ether. The ir-dried precipitte from up to 0.2 ml plsm ws dissolved in 0.5 ml of 10% SDS buffer composed of 0.1 M Tris (hydroxymethyl) minomethne, djusted to ph 7.4, contining 10% (w/v) SDS, 10% (v/v) glycerol, 10% (v/v) 2-mercptoethnol. The tubes were seled nd incubted t room temperture for 18 hr. The smple ws then heted t 100 C for 10 min with vigorous mixing. More thn 99% of the lz5 rdioctivity ws recovered in the combined lipid extrct nd the solubilized plsm. Whole plsm, contining the z5-lbeled lipoproteins, ws prepred for column nlysis by dilution with n equl volume of 10% SDS buffer, heting for 3-5 min t 1Oo"C, nd cooling to room temperture. The rdioctivity in lipid ws determined by direct mesurement or by the difference between delipidted plsm nd whole plsm rdioctivity. Lbeled lipoprotein frctions in 0.15 M NCl/ 2 mm EDTA, ph 7.4, were prepred for column nlysis by dilution with n equl volume of 10% SDS buffer, heting t 100 C for 3-5 min nd cooling to room temperture. Delipidted plsm, whole plsm, or lipoprotein frctions were stble for up to 6 months t -20 C fter dilution with the 10% SDS buffer. Apoprotein seprtion nd nlysis Lbeled poproteins were seprted on 165 X 1.5 cm glss columns (Bio-Rd Lbortories, Richmond, CA) contining Sephrose CL-GB (Phrmci Fine Chemicls, Pisctwy, NJ) equilibrted in column buffer contining 0.1 M sodium phosphte, ph 7.4, nd 1% SDS. Up to 2.0 ml of smple, prepred s described bove, ws pplied under lyer of buffer nd poproteins were eluted 't n verge flow rte of 7-8 mvhr. Frctions were collected t 30-min intervls nd rdiossyed. Columns remined useful for up to 6 months without significnt ltertions of poprotein exclusion volumes. Between 95 nd 100% of pplied rdioctivity ws recovered in the eluted frctions. SDS-polycrylmide gel electrophoresis Following lipid extrction of the lipoproteins, poproteins were dissolved in 1% SDS in 0.1 M sodium phosphte, ph 7.4, nd heted for 2 min t 100 C in the presence of 1 % 2-mercptoethnol. SDS-polycrylmide gel electrophoresis (PAGE) ws crried out in 5 or 10% gels nd the poprotein bnds were identified by stining with Coomssie blue s previously described (23, 24) or by gel slicing nd rdiossy in the cse of 1251-lbeled poproteins. Rdioctivity mesurements Rdiossys of lz51-lbeled lipoproteins nd column frctions of lz5-lbeled poproteins were mde in Serle 1144 gmm scintilltion spectrometer with counting efficiency of 82%. Rdiossys of 3H- or 14C-lbeled lipoproteins were mde in Pckrd Tri-Crb liquid scintilltion spectrometer with n internl stndrd used for quench corrections. Counting errors in llcses were less thn 5%. All results re expressed s 2S.E.M. RESULTS AND DSCUSSON Anlysis of TRL poproteins by SDS-PAGE resolved po C, po E, nd two po B bnds. Clibrtion of SDS-PAGE using moleculr weight stndrds including thyroglobulin (330,000) nd poferritin (220,000), showed the two po B proteins to be between 200,000 nd 400,000 dltons in size corresponding to the rt po B components described by Krishnih, et l. (14). Co-electrophoresed lz5lbeled poproteins were eluted from corresponding unstined gels nd specific 1251-lbeled poproteins were nlyzed by the column procedure which llowed identifiction of poprotein bnds of TRL run by SDScolumns. n Fig. 1, SDS-column nlysis of z5lbeled TRL poproteins present in plsm is shown. The poprotein peks re identified. The clculted vlues for the distribution coefficients (Kd) for the z5-lbeled poproteins is presented in the legend. A moleculr weight clibrtion curve ws constructed using the clculted Kd vlues for stndrd proteins by plotting the logrithm of the moleculr weight versus Kd. From the clibrtion curve, the pproximte moleculr size of the TRL poproteins ws clculted to be: po C = 9600, po E = 37,000, nd po B = 250,000 nd 500,000. We hve designted the po B proteins of higher nd lower moleculr weight po Bh nd po B,. Be- Sprks nd Mrsh SDS-gel chromtogrphy of lipoproteins 515

3 15 t lo 0T 5 L C n 1 J- - L FRACTON NUMBER Fig. 1. Rdioctivity distribution of 12S-lbeled TRL nlyzed by SDS columns where rdioctivity per frction ws plotted ginst frction number. A 20-4 smple of 1z51-lbeled TRL ws dded to 1 ml of rt plsm. A 0.2-ml liquot ws then delipidted nd dissolved in 0.5 ml of 10% SDS buffer s in Methods nd nlyzed on SDS column. Eluted frctions of 3.5 ml were collected every 30 min nd rdiossyed. The peks re lbeled po B (B), po E (E), po C (C), nd - for rdioctive iodide. The first po B pek of higher moleculr weight represents po Bh nd the second po B pek of lower moleculr weight represents po B. The Kd ws clculted by the formul Kd = V, - VdV, - V,. The effluent volume (V,) of mrker substnces ws determined using four different columns nd re s follows; blue dextrn = t 2.4, po Bh = , po B1 = 131.2? 2.1, po E = t 1.5, po C = , 14C-mino cids = , 2-mercptoethnol = t 1.8, z5iodide = , bromophenol blue = The clculted Kd of these mrker substnces ws determined using blue dextrn s the V, nd 2-mercptoethnol s the Vt nd re sfollows; po Bh = 0.054? 0.007, po 3, = t 0.005, po E = , po C = t 0.008, C-mino cids = t 0.004, lz5iodide = , bromophenol blue = W 0 4 m ul m Ow60 FRACTON NUMBER Fig. 2. SDS-Gel filtrtion column chromtogrphy of solubilized TRL poproteins. Protein concentrtion ws mesured by modifiction of the Lowry method (35) nd is plotted s bsorbnce t 660 nm using 1 ml of ech frction in the ssyin column A. Peptide bond bsorbnce t 210 nm is plotted in column B. Both columns re plotted ginst frction number t flow rte of 3.9 ml per 30-min frction. The poprotein peks re mrked po Bh (Bh), po B, (B), po E (E), nd po C (C). A smple contining 2 mg of po-trl ws pplied to ech column in 10% SDS-buffer s described in Methods. TABLE 1. Amino cid composition of Apo Bh nd Apo Bl from rt VLDL Cysteic cid Methionine (sulfoxide) Asprtic cid Threonine Serine Glutmic cid Proline Glycine Alnine Vline soleucine Leucine Tyrosine Phenyllnine Lysine Histidine Arginine Residue.! per 1, The vlues shown re the verges of duplicte nlysis of single preprtion from the pooled serum of 40 rts. Amino cid nlysis ws crried out fter hydrolysis in 6 N HC for 24 hr t 110 C. cuse of the known difficulties of nlyzing the moleculr weight of high moleculr weight hydrophobic proteins in the presence of SDS, we hve chosen not to designte the proteins by moleculr weight until studies of the type crried out by Steele nd Reynolds (30,31) hve been performed. Columns were run on 2 mg of po TRL obtined from the pooled plsm of 50 rts fed d lib nd the poprotein seprtion by SDS-columns is presented in Fig. 2. Ech of the poproteins from the columns ws nlyzed by SDS-5% PAGE nd found to migrte s single bnd corresponding in mobility to the sme poprotein of the originl po TRL. The pure po Bh nd po B1 corresponded to the mobility of the two po B proteins described by Krishnih, et l. (14). Amino cid nlysis ws performed on po Bh nd po B, nd the results re presented in Tble 1. The po Bh nd po B, hd very similr mino cid compositions. The mino cid composition ws lso very similr to rt po LDL (1.006 < d < 1.04 g/m) (32) nd rt po B (14). There ws no tendency for po B ggregtion in the delipidtion of whole plsm s shown in Tble 2. When po TRL ws seprted on columns where the po Bh nd po B, hd Kd s of gin there ws no tendency to ggregte s evidenced by the bsence of higher moleculr weight components. Using the method of Holmquist nd Crlson (33, 34), the po B of lz5-lbeled TRL quntittively precipitted with isopropnol nd the precipitted po B hd the sme column mobility s in the po TRL when nlyzed on SDS-columns. Both po B components were present in in vivo lbeled 516 Journl of Lipid Reserch Volume 22, 1981

4 TABLE 2. Distribution of lbeled TRL poprotein rdioctivity in plsm Preprtion.?.Po B,.4po E.%PO c.4po Bh Pe?cenf lz5-trl" (n = 4) 3.7 f f ? & ? 0.28 z5-trl delipidted (n = 4) 4.1 & f ? f f H-VLDL (n = 3) 19.0? f f f 0.17 Expressed s percent of poprotein rdioctivity. The 1z51-lipid rdioctivity ws mesured independently nd subtrcted from the po C nd totl rdioctivity prior to clcultion. The lipid lbel in z5-trl comigrted with po C nd ws lmost entirely present in phospholipids. n invivo lbeled lipoproteins, some lbel ppers in neutrl lipids tht were extrcted by diethyl ether prior to dissolving the smple in SDS buffer. TRL nd the results re presented in Tble 2 nd Fig. 3. t is unlikely tht the smller po Bh is result of proteolytic ctivity since no peptides were seen in the column nlysis of lbeled TRL nd both peks were present in in vivo lbeled TRL isolted in the presence of protese inhibitor (toluene sulfonyl fluoride) nd zide. n ddition to the nlysis of poproteins, the present method llows for the mesurement of lbeled peptides nd mino cids which seprte from lz5-10" P' e -- N 0 - X 5 V 4 " B B 2 -- u, 11 tf u L FRACTON NUMBER Fig. 3. Rdioctivity distribution of 14C-lbeled TRL nlyzed by SDS columns. Rdioctive 14C-lbeled TRL ws prepred from sucrose-fed rts fed d lib injected with 14C-lbeled mino cids, s discussed in Methods. The ultrcentrifuglly isolted "C-lbeled TRL frction ws dded to n equl volume of 10% SDS buffer nd pplied to the SDS column. Frctions of 3.5 ml were collected every 30 min, rdiossyed, nd the rdioctivity per frction ws plotted ginst frction number. The poprotein bnds re indicted bove ech pek s in Fig. 1. iodide in study of degrdtion of lz5-lbeled poproteins. Both lz5iodide nd bromophenol blue hd elution volumes greter thn totl volume indicting interction with the grose gel s shown in Fig. 1. There ws consistent contmintion of ll preprtions of lz5-lbeled TRL in spite of extensive dilysis with lz5iodide rdioctivity (2-5% of the totl rdioctivity) nd this iodide could be mesured reproducibly by SDS-columns. The SDS-columns offer the dvntge of complete rdioctivity recovery even in the presence of plsm proteins. The disdvntge of whole plsm nlysis is the inbility to determine individul poprotein specific ctivity in ech lipoprotein frction. n ddition, complete resolution of po E from po A- nd po A-V is not possible in smples contining these poproteins. The low moleculr weight po C proteins seprte s group. The individul plsm lipoprotein density clsses s isolted by ultrcentrifugtion cn be redily frctionted by SDS-columns. Seprtion of unlbeled SDS-solubilized TRL poproteins by SDS-columns is presented in Fig. 2. The protein mesurement is mde by modifiction of the method of Lowry, et l. (35) or by bsorbnce t 210 nm. n nlysis of lbeled proteins, the rdioctivity is mesured nd poprotein specific ctivity is clculted. n smples contining po A-, po A-V, nd po E, the column frctions contining these poproteins cn be pooled nd the individul poprotein specific ctivities determined by SDS-PAGE nlysis. ndividul po C protein specific ctivity mesurement requires nlyticl isoelectric focusing. The use of SDS-columns hs improved the bility to ccount for ll rdioctivity in smples obtined from metbolic experiments, but must be supplemented by other methods, s indicted, to obtin specific ctivities of ll of the popr0teins. We wish to thnk Arline Ritz, Bruce LMont, Srh Mssey, nd Oleh Hntiuk for their excellent technicl ssistnce. We re prticulrly indebted to Dr. Joel Rosenbloom for Spurks nd Mrsh SDS-gel chromtogrphy of lipoproteins 517

5 the mino cid nlyses. This work ws supported in prt serum polipoproteins by isoelectric focusing. 11. by N..H. Grnt No. Pol-HL Studies on Mnuscript received 8 August 1980 nd in revised form 14 November REFERENCES 1. McFrlne, A. S Efficient trce-lbeling of proteins with iodine. Nture. 182: Bilheimer, D. W., S. Eisenberg, nd R.. Levy, The metbolism of very low density lipoproteins. 1. Preliminry in vitro nd in vivo observtions. Biochim. Biophys. Act. 260: Fidge, N. H A review of methods nd metbolic studies ssocited with the rdioiodintion of lipoproteins. Clin. Chim. Act. 52: Fidge, N. H Studies on the rdioiodintion of very low density lipoproteins obtined from different mmmlin species. Clin. Chim. Act. 52: Eisenberg, S., nd R.. Levy Lipoprotein metbolism. Adv. Lipid Res. 13: Schefer, E. J., S. Eisenberg, nd R.. Levy Lipoprotein poprotein metbo1ism.j. Lipid Res. 19: Biermn, E. L., 0. Stein, nd Y. Stein Lipoprotein uptke nd metbolism by ortic smooth muscle cells in culture. Circ. Res. 35: Kne, J. P A rpid electrophoretic technique for identifiction of subunit species of poproteins in serum lipoproteins. Anl. Biochem. 53: Mhley, R. W., nd K. S. Holcombe Altertions of the plsm lipoproteins following cholesterol feeding in the rt. J. Lipid Res. 18: Finru, M., R. J. Hvel, nd K. mizumi Apoprotein content of plsm lipoproteins of the rt seprted by gel chromtogrphy or ultrcentrifugtion. Biochem. Med. 17: Scnu, A. M., nd C. Edelstein Solubility in queous solutions of ethnol of the smll moleculr weight peptides of the serum very low density nd high density lipoproteins. Anl. Biochem. 44: Fergemn, O., T. St, J. P. Kne, nd R. J. Hvel Metbolism of poprotein B of plsm very low density lipoproteins in the rt. j. Clin. nvest. 56: Kne, J. P., D.A. Hrdmn, nd H. E. Pulus Heterogeneity of polipoprotein B: isoltion of new species from humn chylomicrons. Proc. Ntl. Acd. Sci. USA. 77: Krishnih, K. V., L. F. Wlker, J. Borensztin, G. Schonfeld, nd G. S. Getz Apolipoprotein B vrint derived from rt intestine. Proc. Ntl. Acd. Sci. USA. 77: Weber, K., J. R. Pringle, nd M. Osborn Mesurement of moleculr weights by electrophoresis on SDScrylmide gel. n Methods in Enzymology. Vol. X, XV. C. H. W. Hirs nd S. N. Timshesi, editors. Acdemic Press, New York Swney, J. B., H. Reese, nd H. A. Eder Polypeptide composition of rt high density lipoprotein: chrcteriztion by SDS-gel electrophoresis. Biochem. Biophys. Res. Commun. 59: Gidez, L.., J. B. Swney, nd S. Murnne Anlysis of rt serum polipoproteins by isoelectric focusing.. Studies on the middle moleculr weight subunits of HDL nd VLDL. J. Lipid Res. 18: Swney, J. B., nd L.. Gidez Anlysis of rt 518 Journl of Lipid Reserch Volume 22, 1981 the low moleculr weight subunits. J. Lipid Res. 18: Hglund, H soelectric focusingin ph grdients. A technique for frctiontion nd c6r cteaztion of mpholytes. Methods Biochem. Anl. 19: Albers, J. J., nd A. M. Scnu soelectric frctiontion nd chrcteriztion of polypeptides from humn serum very low density lipoproteins. Biochm. Biophys. Act. 236: Albers, J. J., V. Albers, nd F. Aldjem soelectric heterogeneity of the mjor polypeptide of humn serum high density lipoproteins. Biochem. Med. 5: Edelstein, C., C. T. Lim, nd A. M. Scnu The serum high density lipoproteins of Mccus rhesus. 11. soltion, purifiction, nd chrcteriztion of their two mjor polypeptides. J. Biol. Chem. 24%: Mrsh, J. B., nd C. E. Sprks Heptic secretion of lipoproteins in the rt nd the effect of experimentl nephrosis. J. Clin. nvest. 64: Mrsh, J. B., nd C. E. Sprks Lipoproteins in experimentl nephrosis: plsm levels nd composition. Metbolism. 28: Dy, C. E., nd R. S. Levy Determintion of the moleculr weight of poprotein subunits from low density lipoproteins by gel filtrtion. J. Lipid Res. 9: Brown, W. V., R.. Levy, nd D. S. Fredrickson Studies of the proteins in humn plsm very low density lipoproteins. J. Biol. Ch. 244: Mrsh, J Apoproteins of the lipoproteins in nonrecirculting perfuste of rt liver. J. Lipid Res. 17: Cpuzzi, D. M., C. E. Sprks, nd J. L. DeHoff Effect of residul enzymes on degrdtion of rdioiodinted VLDL by collgense-dispersed heptocytes. Biochem. Biophys. Res. Commun. 90: Lux, S. E., K. M. John, nd H. B. Brewer soltion nd chrcteriztion of po Lp-Gln ii (Apo A-), plsm high density polipoprotein contining two identicl polypeptide chins. J. Biol. Ch. 247: Steele, J. C. H., Jr., nd J. A. Reynolds Chrcteriztion of the polipoprotein B polypeptide of humn plsm low density lipoprotein in detergent nd denturnt solutions. J. Biol. Chem. 254: Steele, J. C. H. Jr., nd J. A. Reynolds Moleculr weight nd hydrodynmic properties of polipoprotein B in gunidine hydrochloride nd sodium dodecyl sulfte so1utions.j. Biol. Chem. 254: Kog, S., D. L. Horwitz, nd A. M. Scnu soltion nd properties of lipoproteins from norml rt serum. J. Lipid Res. 10: Holmquist, L., nd K. Crlson Selective extrction of humn serum very low density polipoproteins with orgnic solvents. Biochem. Biophys. Act. 493: Holmquist, L., K. Crlson, nd L. A. Crlson Comprison between the use of isopropnol nd tetrmethylure for the solubiliztion nd quntittion of humn serum very low density polipoproteins. Anl. Biochem. 88: Lowry, 0. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Fohn phenol regent. J. Biol. Chem. 193: