AMINO-ACID COMPOSITION OF SERUM PROTEINS

Transcription

1 J. clin. Pth. (1955), 8, 153. AMINOACID COMPOSITION OF SERM PROTEINS IN HEALTH AND DISEASE BY A. J. CMMINGS AND F. V. FLYNN From the Deprtment of Clinicl Pthology, niversity College Hospitl, London Electrophoresis of serum proteins in disese frequently discloses bnormlities. The ltertions re rrely dignostic; thus diffuse increse of the yglobulin is common to mny cliniclly unrelted conditions. It would be of considerble vlue therefore if it were possible to distinguish between the proteins which re responsible for common bnormlity in the electrophoretic pttern. It ws with view to relting the composition of the serum proteins to disese tht we hve undertken study of the minocid composition of the electrophoretic frctions in liver disese, kidney disese, nd myelomtosis, conditions in which it is well known tht the serum proteins show gret bnormlity. Existing dt on the minocid composition of serum proteins re very megre, nd we hve been unble to find ny literture deling with the minocid content of electrophoreticlly pure serum protein frctions. The composition of the plsm proteins in helthy subjects hs been investigted by Brnd, Kssel], nd Sidel (1944), Shemin (1945), Brnd (194), nd Brnd nd Edsll (1947), who give dt for the percentge composition of certin minocids in lbumin nd vrious globulin frctions seprted by precipittion methods. Brnd nd Edsll (1947) include results of complete minocid nlyses of nd 5globulins s well s of lbumin nd yglobulin, but the frctions were not electrophoreticlly homogeneous. The findings hve been reviewed by Mrrck nd Hoch (1949). The content of few minocids in plsm proteins hs been studied in pthologicl conditions. Tuchmn nd Sobotk (1932) found discrepncies in Wu nd Kjeldhl methods of serum protein determintion in cses with nephrosis or nephritis with oedem, nd concluded tht in these conditions the lbumin tyrosine content is incresed while the globulin tyrosine is diminished. Alving nd Mirsky (193) studied the cystine content of the plsm proteins in eight cses of kidney disese nd in five helthy individuls; prt of the lbumin frction from ptients with the nephrotic syndrome ws L (RECEIVED FOR PBLICATION OCTOBER 8, 1954) found to hve n bnormlly low cystine content. Blint nd Bilint (1942 nd 1943) estimted tyrosine, tryptophn, cystine, rginine, nd histidine in the plsm proteins from ptients with cute infections nd in cses of nemi. In cute infections they found tyrosine nd tryptophn decresed, nd cystine incresed, in the lbumin, nd the reverse chnges in the globulin. Emmrich (195) investigted the cystine content of norml nd pthologicl serum proteins nd found bnorml levels in protein deficiency, cirrhosis of the liver, chronic kidney disese, tuberculosis, nd other chronic inflmmtory processes. Schuck (195) found vrition in the cystine nd methionine content of the plsm proteins in pregnncy with bnorml levels in the toxemiofpregnncy. Muiting(1951),who estimted only methionine, showed tht the level of this minocid in serum protein ws lower thn norml in prenchymtous liver disese nd high in cses of the nephrotic syndrome. Schreier (1951) estimted biologiclly threonine, vline, leucine, isoleucine, phenyllnine, lysine, histidine, cystine, nd methionine in serum lbumin from four cses of the nephrotic syndrome nd single cses of liver disese, Hodgkin's disese, leukemi, nd crcinomtosis, but found no significnt vrition from norml. Clinicl Mteril Dt re presented here of the composition of the serum proteins of six helthy young dults, six ptients with the nephrotic syndrome, six with liver disese, nd six with multiple myelom. Of the ptients suffering from liver disese, four hd cirrhosis, one hd subcute virl heptitis, nd one ws cse of cute heptic necrosis. Except for one ptient with the nephrotic syndrome nd one with cirrhosis, ll the ptients were dults, nd ll except two were ttending niversity College Hospitl. In ech cse the clinicl fetures of the condition were typicl nd the dignosis ws supported by pproprite pthologicl investigtions or confirmed t necropsy. The ser from ll the J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from on 14 August 218 by guest. Protected by copyright.

2 154 ptients showed bnormlities in the electrophoretic pttern of the proteins; notbly there ws n increse of the yglobulin nd decrese of the lbumin in the cses of liver disese, n increse of the C2globulin nd decrese of the lbumin nd yglobulin in those with the nephrotic syndrome, nd mrked compct increse of the yglobulin in the cses with myelom. A. J. CMMINGS nd F. V. FL YNN Method The proteins were frctionted by electrophoresis on filter pper, the frctions hydrolysed, their constituent minocids seprted nd identified by pper chromtogrphy nd then estimted colorimetriclly. The,globulin ws not nlysed becuse insufficient ws vilble. Electrophoresis.The proteins were frctionted by pper electrophoresis s described by Flynn nd de Myo (1951). Serum,.2 ml., ws pplied to ech of eight or 1 filter pper strips (Whtmn No. 31 extr thick) nd potentil of 12 volts pplied for 18 hours. The strips were heted t 8 C. until just dry before stining with bromphenolblue. Isoltion of Proteirs.The protein frctions so detected were cut out nd steeped in pproximtely 15 ml. of.5 Nsodium hydroxide for 24 hours with occsionl shking. The solutions were filtered, nd the filtrte djusted to ph 5 with sulphuric cid. Mercuric chloride, I g., ws dded nd the whole heted until just boiling. The cogulted proteins were llowed to sediment for 24 hours nd the supemtnt fluid decnted s completely s possible. The remining sludge ws trnsferred to centrifuge tube, being wshed in with wter, nd the proteins collected by centrifuging; they were then wshed twice by mixing with wter nd recentrifuging. Preprtion of the Hydrolyste.To the protein in ech centrifuge tube 5 ml. of Nhydrochloric cid ws dded. The.tube ws seled nd incubted t 1 C. for 15 hours. The hydrolyste ws then evported to dryness under reduced pressure nd the residue dissolved in 2 ml. of 1% (v/v) isopropyl lcohol s recommended by Block (195). This solution ws deslted electrolyticlly nd subsequently smple tken for totl nitrogen determintion by microkjeldhl procedure. Chromtogrphy.Twowy chromtogrphy ws crried out s described by Dent (1948) using Whtmn No. 4 filter pper. A volume of hydrolyste equivlent to.1 mg. of nitrogen ws pplied to ech chromtogrm. The developed chromtogrms were lightly spryed with.2% (v/v) ninhydrin in nbutnol nd llowed to stnd t room temperture until the colour developed; usully they were left overnight. Determintion of Aminocids.From ech chromtogrm the minocid spots were cut out with mrgin of unstined pper, together with pper blnks of the sme sie. In prctice ll the minocid spots could often be contined within circles of 1+ in. dimeter nd then single blnk of the sme dimensions would be used. The cutout minocid spots were trnsferred to testtubes nd 1 ml. of 5% (v/v) ninhydrin in 9% isopropyl lcohol (Block, 195) dded to ech. The tubes were shken well nd incubted in wter bth t 8 C. for 1 minutes. After cooling, 5 ml. of 7% (v/v) cetone in wter ws dded to ech tube. The tubes were shken to dissolve the dye nd then stood for 2 minutes to complete the solution (Fowden, 1951). Certin of the minocids gve too intense colour for convenient reding in the photometer; in these instnces further dilution with 7% cetone ws crried out. The opticl densities were mesured t 57 m,4. The chromtogrphy nd minocid estimtions were crried out in duplicte. Isoleucine nd leucine were estimted together, since their seprtion cnnot be effected with the solvents used. Proline ws not estimted s the recovery ws very poor, nd in the cse of cystine nd other minocids the mount ws too smll to determine. Clcultion.Fctors were obtined correlting the colorimetric redings with the mounts of individul minocids present by subjecting proteins of known composition to the bove procedure. Csein, geltine nd electrophoreticlly pure norml serum lbumin were used for this purpose. The dt given by Hwk, Oser, nd Summerson (1947) for csein nd geltine, nd by Brnd nd Edsll (1947) for serum lbumin, were used for the clcultion of the fctors; the dt given by these uthors were first corrected to express the percentge minocid composition per 1 g. minocids. By this method of clcultion llownce is mde for vritions in the mount of colour produced in the rection with ninhydrin, s well s for ny lck of quntittive recovery of the individul minocids. The colorimetric redings were multiplied by the pproprite fctors nd the vlues thus obtined for the ctul mounts of individul minocids present were then expressed s percentge of the totl mount of minocids estimted. Reproducibility.The finl figure for ech individul minocid vlue ws shown to be reproducible to within 1% for ll the cids except lysine nd histidine, which were reproducible to within 15%, nd for rginine which ws reproducible to within 2%. The mount of rginine detected ppers to be relted to the length of time required for deslting; if this is prolonged rginine is converted to ornithine (Stein nd Moore, 1951). Results The results re shown in Figs. 112, nd vlues for the verge composition of the norml serum proteins re given in Tble 1. Tble I shows tht the different frctions differ somewht in composition, the content of serine, lnine, threonine, nd lysine being prticulrly vrible. These findings re in ccord with those of other uthors using different methods of nlysis (Brnd nd Edsll, 1947). Anlysis of the results from the pthologicl ser shows tht in no cse re the serum proteins grossly J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from on 14 August 218 by guest. Protected by copyright.

3 ':3 43 J '3 j I i C: F cl LA : LLJ u t3 ZJ Z J. J >. CA j 1 i ẕ j > L Z Z Z Z CA 2 ZO Z I > I J C H F Zn I o FIG. I J u L i Z Z Z Z L I CL V) I > I j : Zj _. _ > J uj > j J Z u Z Z * J > i J '3 L CLi J 3j j i In FIG. 2 I w ul i ZZZ Z u j I i LL, u. Z Z Z Z Z > ne I I V) IC %' J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from FIG. 3 IAGo. 4 FiGs. 14.Aminocid composition of serum protem frctions in six cses of prenchymtous liver disese. Vlues expressed's' g. per 1 g. minocid estimted. Shded re represents rnge of vlues found in six normls. Fig. I is for lbumin, Fig. 2 for,.globulin (five cses only), Fig. 3 for flglobulin, nd Fig. 4 for vglobulin. on 14 August 218 by guest. Protected by copyright.

4 uj 1 Z Z' LL, F Z Z 27i J Q l Zu Z LLI cic F Z Z D D J uj >. " j J : Ji > J j LJ LLJ Z J 2 Z J Z O Z Z Z LI cl l R I O LA, ( ~~~ 8 ~~~~~~~~ Z Z Z L _~~ ~ FIG. 5 FIG. L j LJ Z Z Z Z u Z 3 Z ~ ~ > I > I j F F I D j S j VI CL (A Z w u L2 u Z Z Z Z J ' Z. I J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from FIG. 7 FIG. 8 FIGS. 58.Aminocid composition of serum protein frctions in six cses of nephrotic syndrome. Vlues expressed s g. per 1 g. minocid estimted. Shded re represents rnge of vlues found in six normls. Fig. 5 is for lbumin, Fig. for 2globulin, Fig. 7 for,globulin, nd Fig. 8 for yglobulin. on 14 August 218 by guest. Protected by copyright.

5 1 > ), J J * l J J u Z C uf LLi ) Z r _L J > jz Z o Z T1 ~~ u Z L Z J > i FiG. 9 FIG. 1 In * ~ Z. (A > LA > % vo FIG. 11 FIG. 12 i Z Z Z Z2l ull I 2 W Z Z Z l 1) 2 I5 )w p %' FIGS. 912.Aminocid composition of serum protein frctions in six cses of multiple myelom. Vlues expressed s g. per 1 g. minocid estimted. Shded re represents rnge of vlues found in six normls. Fig. 9 is for lbumin, Fig. 1 for 2globulin, Fig. 11 for j3globulin, nd Fig. 12 for yglobulin. 1 7 J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from on 14 August 218 by guest. Protected by copyright.

6 158 TABLE I MEAN VAES* FOR THE COMPOSITION OF NORMAL SERM PROTEINS Albumin 2globulin,globulin yglobulin Glutmic cid Leucine Alnine Lysine Asprtic cid Vline Phenyllnine Tyrosine Arginine Threonine Serine Histidine Glycine * Vlues expressed s g. per 1 g. minocid estimted. bnorml in composition, t lest in terms of those minocids estimted. More criticl exmintion of the dt, however, shows tht minor vritions my occur in individul cses. In Tble 1I the frequency nd direction of devitions from the norml rnge which re considered to be definitely outside the rnge of experimentl error re indicted. Discussion Pper electrophoresis of proteins hs severl dvntges over the clssicl Tiselius technique, n importnt one being tht ll the frctions re entirely seprted from one nother in single opertion. This technique therefore provided us with simple method of isolting electrophoreticlly pure protein frctions for the purposes of this study. Abnormlity of the chemicl composition of the serum proteins might be expected in disese, since the proteins often show considerble vrition from norml in their physicl properties. Some of these chnges, however, my result from n ltertion in the structure of the protein molecules rther thn Leucine A. J. CMMINGS nd F. V. FLYNN from n bnorml chemicl composition. A recent report shows tht gret devition from norml my occur in the chemicl composition of proteins under rtificil circumstnces: thus, in infnts fed on lysinedeficient diets, proteins ppered in which lysine ws replced by rginine (Lncet, 1953). Our findings estblish tht there is no gross devition in the proportions of t lest 13 minocids in the serum proteins, even when there is mrked disturbnce of protein metbolism. The minor chnges we hve found must be interpreted with cution. The fct tht dt re vilble from only six normls must be kept in mind, nd it is this in prticulr tht hs prevented sttisticl nlysis of the results. It must lso be noted tht when the percentge of one minocid is incresed the increse occurs t the expense of others which must therefore be decresed. The experimentl error is regrettbly lrge nd indeed estimtion of some of the minocids present in smller quntity ws bndoned becuse of this; however, s noted in Tble II, some of the results fll well outside this error. Despite ll these reservtions our findings in the pthologicl ser require some comment. The results.show more frequent devitions from norml composition in the cses of liver disese thn in the other conditions studied. It is lso of interest tht in liver disese nd myelomtosis bnormlities occurred most often in the yglobulin frction, which ws the frction showing the gretest increse in concentrtion. In the cses of the nephrotic syndrome, however, lthough the 2globulin showed the gretest increse, bnormlities of composition were found more frequently in the (globulin. Vritions in the composition of the serum protein frctions in disese my result either from the production of proteins of bnorml composition or TABLE IX FREQENCY OF DEVIATION OF AMINOACID COMPOSITION FROM THE NORMAL RANGE Liver Disese Kidney Disese Multiple Myelom Albumin s2 i Y Albumin 2 i Albuminl 2 Y Alnine Asprtic cid.... t 1_ _ t Phenyllnine... 1 t t 1 _ t 4 t t tl1t Tyrosine T TI Tit t T_t Threonine l _ _ Serine Glycine t tti t = Increse nd 4 =decrese in concentrtion. Number of rrows corresponds to number of cses showing bnormlity. J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from on 14 August 218 by guest. Protected by copyright.

7 AMINOACID COMPOSITION OF SERM PROTEINS from ltertion in the reltive proportions of proteins normlly present. With regrd to the ltter possibility it is probble tht mny different proteins together mke up the electrophoretic spectrum of serum, nd since the mjor bnds of the spectrum show differences in chemicl composition it is likely tht the different proteins which together constitute prticulr frction will lso differ slightly in composition. If this is so, then mrked increse or decrese of the concentrtion of prticulr component protein could cuse the overll composition of tht frction to differ from norml. Vrition in the proportion of component proteins could be held responsible for the smll vrition in composition found in the serum proteins of helthy individuls. In ll the cses of liver disese studied, electrophoresis showed diffuse increse of the serum rglobulins, the increse ffecting the fster moving y,globulins to, the gretest extent. The chemicl bnormlities found in the whole frction might therefore be due to selective increse in the y,globulins. On the other hnd this finding, nd the bnormlities found in the other frctions, could be the result of synthesis of bnorml proteins. In the nephrotic syndrome the predominnt proteins to pper in the urine re lbumin nd ocr nd (globulins (Slter nd Kunkel, 1953; Squire, 1953; Flynn, 1954). This differentil loss of protein cn be relted to the moleculr weight of the proteins (Squire, 1953), nd my serve to explin why we encountered vrition in protein composition most frequently in the,8globulin. The norml serum (globulin frction is composed of two min groups of proteins, one with moleculr weight of round 9,, the other with moleculr weight of bout 1,3,. Differentil loss of the lower moleculr weight component, should it differ somewht in minocid composition from tht of the other component, could then be responsible for the serum,8globulin ppering bnorml in composition. Lck of bnormlity in the serum lbumin could be ttributed to ll the lbumin components pssing into the urine with equl fcility. The findings of Albnese, Dvis, Smetk, nd Lein (1949) suggested tht the lbumin lost in the urine in the nephrotic syndrome my differ in composition from norml plsm lbumin. Their results, tken in conjunction with our finding tht the serum lbumin is norml in composition, suggest tht bnorml lbumins re synthesied but re immeditely excreted in the urine. However, it must be noted tht Schreier's (1951) results on the urine from two cses do not confirm the findings of Albnese et l. (1949). In myelomtosis the presence of chemiclly bnorml serum protein might well hve been expected in view of the findings of Dent nd Rose (1949) tht BenceJones protein entirely lcks methionine. However, our findings with 13 minocids show little ltertion in the composition of the yglobulin frction despite its gret increse in concentrtion. Tking ll the bove results into considertion one my conclude tht minocid nlysis of serum protein frctions is unlikely to be of dignostic vlue. Summry The minocid composition of electrophoreticlly homogeneous serum protein frctions hs been studied in six helthy young dults nd in six cses ech of liver disese, the nephrotic syndrome, nd myelomtosis. Thirteen minocids were estimted quntittively. No gross or chrcteristic ltertion from norml ws found in the pthologicl ser in the lbumin, 2(P, nd yglobulins. Minor devitions from norml composition were found in individul cses. The possible significnce of these findings is discussed. Work included here ws crried out during the tenure by one of us (A. J. C.) of grnt from the Rockefeller Reserch Fund of niversity College Hospitl Medicl School. We wish to thnk Professor M. Miels nd Dr. C. E. Dent for helpful criticism nd dvice. REFERENCES Albnese, A. A., Dvis, V. I., Smetk, E. M., nd Lein, M. (1949). J. Lb. clin. Med., 34, 32. Alving, A. S., nd Mirsky, A. E. (193). J. clin. Invest., 15, 215. Blint, P., nd Bilint, M. (1942). Biochem. Z., 313, 192. (1943). Ibid., 315, 41. Block, R. J. (195). Anlyt. Chem., 22, Brnd, E. (194). Ann. N.Y. Acd. Sci., 47, 187. nd Edsll, J. T. (1947). Ann. Rev. Biochem., 1, 223. Kssell, B., nd Sidel, L. J. (1944). J. clin. Invest., 23, 437. Dent, C. E. (1948). Biochem. J., 43, 19. nd Rose, G. A. (1949). Ibid., 44, 1. Emmrich, R. (195). Z. ges. inn. Med., 5, 42. Flynn, F. V. (1954). Proc. roy. Soc. Med., 47, 827. nd de Myo, P. (1951). Lncet, 2, 235. Fowden, L. (1951). Biochem. J., 48, 327. Hwk, P. B., Oser, B. L., nd Summerson, W. H. (1947). Prcticl Physiologicl Chemistry, 12th ed. Churchill, London. Lncet, 1953, 2, Mrrck, J. R., nd Hoch, H. (1949). Journl of Clinicl Pthology, 2, 11. Muting, D. (1951). Z. klin. Med., 147, 478. Schreier, K. (1951). Biochem. Z., 321, 528. Schuck, J. (195). Arch. Gynk., 178, 217. Shemin, D. (1945). J. biol. Chem., 159, Slter, R. J., nd Kunkel, H. G. (1953). J. Lb. clin. Med., 41, 19. Squire, J. R. (1953). Brit. med. J., 2, Stein, W. H., nd Moore, S. (1951). J. biol. Chem., 19, 13. Tuchmn, L. R., nd Sobotk, H. (1932). Ibid., 98, 35. J Clin Pthol: first published s 1.113/jcp on 1 My Downloded from on 14 August 218 by guest. Protected by copyright.