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Characterization of the electrophoretic components of the sera of dog, rat and man in terms of six amino acids
Characterization of the Electrophoretic Components of the Sera of Dog, Rat and Man in Terms of Six Amino Acids Julius Schultz, George Grannis, Hazel Kimmel and Harry Shay From the Samuel S. Fels Research Institute, Temple University Philadelphia, Pennsylvania Received
January
School of Medicine,
3, 1955
INTRODUCTION Zone electrophoresis of animal sera carried out according to the techniques of Kunkel and Slater (1) made possible the preparation of the classical Tiselius electrophoret,ic components of the dog, rat, and man as described in a previous report from this laboratory (2). The possibility that such protein preparations may be identified by means other than mobility was suggested by the data reported by Brand and Edsall (3) on the amino acid composition of some of the proteins of human sera isolated by chemical fractionation procedures. Amino acid paper chromatography of the hydrolyzates of such readily prepared proteins offered an available means for establishing criteria for identification of electrophoretic components that could be used to study changes in protein composition of the major Tiselius groups. This was not possible in terms depending on the quantitative variation in the area of a given range of mobility. Such findings could be applied to any protein mixture that can be separated by zone electrophoresis. In a preliminary report of this study (4, 5), amino acid hydrolyzates of the serum proteins of the rat separated either on paper or starch yielded amino acid chromatograms that showed certain features characteristic of each of the groups of proteins whose mobility fell within major groups of the classical Tiselius pattern. The present report deals with the quantitative aspects of these findings, which have been extended to three animal species, and show that not only are each of the protein fractions of a given species distinct from the other components of the same serum, but that there is a remarkable 174
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similarity between analogous components from one species to the next. This means that the distribution of the amino acids amongst the serum proteins of the normal dog, rat, and man is similar, and that the same amino acids are the characteristic features of a given prot,ein group in t.he three species. The criteria for the identification of the serum proteins are established by considering the electrophoretic pattern to result from a system of proteins whose amino acids constitute a definitive distribution in relation to their electrophoretic mobility. The mobility of any protein, ot)hel than the normal serum proteins found in the serum would not necessarily be related in terms of its own amino acids to the same definitive distribution reported here for the serum proteins. METHODS The methods for the preparation of the protein fractions used in these expcriments have been reported previously (2). In preliminary studies there was reasorlable evidence that glutamic acid, aspartic acid, alanine, glycine, serine and thrconine, which represent two dicarboxylic acids, tFo straight-chain aliphatic acids, and two hydroxglated aliphatic acids, could serve as a good basis for comparing the serum proteins. One decided advantage, besides representing some of the kc,> groups of amino acids, n-as that they could be readily analyzed in a routine fashion as a single streak wherein each amino acid appeared as a single spot. In this R-a: replicate analyses of a single hydrolyzate as recommended by Block et al. (6) were carried out with ease. A given series of streaks were joined together for reading in an automatic densitometer attached to an automatic recorder. Other methods depending on column chromatographic separation were not suitable both from the time required for a given analysis, and the small amount of material available in most cases. For example, after electrophoresis of as little as 0.2-0.4 ml. of serum of a single rat the proteins could be separated and hydrolyzed to yield sufficient material for repeated analysis of amino acids that were suitable to diffrrentiatts the proteins (5).
Hydrolysis H&o~!Js~s and Preparation of Hydrolyzates Jar Amino Acid (‘hrotilatogrclpl1.y. Aliquots of albumin and in most cases the entire globulin fractions were hyclroIyzed in G S HCI for 24 hr. at 100°C. Experiments with trifluoroacetic acigl r+ sulted iI large losses of threonine and serine heretofore not reported l)y others. Treatment of synthetic hydrolyzates of serum albumin 01 r-globulin with trifluoroacetic acid sho~~tl on chromatography distinct losses of these two amino acids. Removal of the IICl with chloroform solutions of di~2~eth~lhexylarnine (Gj did not yield preparations suitable for the chromatographic solvents used in these studies. Direct removal of the acid by evaporation in uacuo, and solution of the residue, iI1 10% isopropyl alcohol proved most satisfactory.
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Chromatography The solvent system used for quantitative amino acid paper chromatography of aspartic acid, glutamic acid, glycine, serine, threonine, and alanine was phenolwater buffered at pH 12, as described by McFarren and Mills (7)~. Acetone added to the butanol-ninhydrin reagent to increase the rate of evaporation of the solvent after color development (8), and precauticns taken to prevent heating the papers in the presence of phenol aided materhlly in obtaining well-developed spots with a minimum of tailing. In some of our experiments the chromatograms were run in ll>s X 435 X 12 in. museum jars in which the paper was suspended by all-glass supports. In most cases, however, a commercial “Chromatocab” was used, which gave more consistent results because >f better control of temperature and humidity. Simultaneous determination of a greater number of samples could be run on a single sheet of paper. In a sample run, the initial concentration of a-amino N was determined on the hydrolyzate by the ninhydrin method proposed by Troll and Cannan (9). From 1 to 10 ~1. of hydrolyzate was placed alternately with standard spots across the bottom of the llaper, so that up to 22 spots which included 11 unknowns were chromatographetl together. From 1 to 4 such analyses were averaged in determining the values fcr a-amino N for each of the amino acids reported here. A number of samples of leaper (Whatman No. 1) were tested by placing 22 spots of the same standard acrok,s the paper, and the density was read after color development. Deviations noted in certain parts of the paper were taken into consideration when adjacent spots due to standard and unknown were found to be “off” the curve which showed the relationship between density versus the logarithm of a-amino N concentratior: . Block et al. (6) suggested that standard hydrolyzates be used which were close in relative amino acid composition to that of the unknown. The standards used in these experiments were therefore made up according to the amino acid composition of human serum albumin, and a-, ,8-, and r-globulin reported by Brand. We have found that only when an analogous component was used as standard in this way, e.g., albumin read against albumin, and globulin against the corresponding globulin, were reproducible results obtained. Another difficulty that was encountered involved the marked variation in concentration of two amino acids such as glycine and glutamic acid of albumin. Here in the lowest concentrations, glutamic acid gives dense spots, while glycine is hardly detectable; as one increases the concentration, the glycine reaches suitable densities only at a point where the glutamic acid spot is too intense to include in the analysis. It is in such cases that the replicate series and repeated runs become necessary.
Calculation of Results The total a-amino N of the six amino acids w-as determined, and the amount of each amino acid was expressed as a percentage of this value for each hydrolyzate analyzed. The terms used are thus mole fraction per cent of the total moles of amino acid represented by the aspartic, glutamic, serine, glycine, threonine, and alanine, of a given protein fraction. In this way errors other than those due to the amino acid determination are reduced to a minimum; for the nintydrin method of Troll and Cannan was found to be useful as a guide only, and the Lowry et al. (10)
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acaid values. The variation between species can be seen to be less than that, of the same component within a given species. In Fig. 1, examples of typical chromatograms of the hydrolyzates of the serum proteins of the three species are presented. Certjnin generalities, rwdily drawn from the data of Table I, (WI be observed with th(l naked cyc from these chromatograms. For example, the albumins of wdh species are characterized by the intense ulaninc~ spot when corn pared to the threonine spot. of the same streak; this relation iti not tho santc: in the, streaks of t,he other components. In fact, t’hctr-globulins she\\ more iutnwc thrconine spotIs than alanine, while in t hr CILSUof the @globulins the wversc is seen; t,hus differentiat~ing uot only t hew tjwo :~d,jacw~t~mobility areas, but also the albumin. ‘I’hc glycine-serine spots .W~IX: IO determine the albumin and al-globulins ill the samfb n1au11~~r. .111d nhcll one considers the fluctuations in iutjrnsity, ocz-globulins (WI Iw sew to be different in relative composition from the al- a11d/3-glotw lilts. III o\lr labornt80ry those working with t.hesc c~hromat,ograms h:lr~~ lcwned to guess accurately at the origin of a giver1 streak \vith grwt, rcgularitv. L\lthough we recognize t’hat such obscrvatious arc s~~~)~jwti\~e, w \\-ish to present them as a means of a readily :~vail:~ble clllalitativr~ Iwt of the I1ature of a mixture of proteins from a (wnimoi1 sours that lirr) intlic~ntc the points of application of 3--4 ~1. of synthetic 11ydrolyz:~tc~s corr~w I)nntlirlg to human wrum proteins using the data of Brand and Edsnll (R!. This wl)rwcnt~s :i I otal of 4 pg. of a-amino N that was placed at each origin. Sinw no (131:~ on alaninc~ ~\~re available, this amino acid was eliminatetl from t,hcl h!-clrolJ.z:~t~s, c~src~l)~ for albumin where t,he value for bovine serum albumin \xas usfIll. l’hf> chromatograms were run in buffered phenol at pH 12.0 as descrilwl I)!- 111*F’arR-II :~rr(l Rlills (7) \vith t,hc exception that ascending technique NW use~l. Toil that tlw chnr:rct(lr of each streak is distinct from the other; e.g., t,hc~ low glycirw of nll)umin, thtt high glycine of &globulin and the high serinr of -,-globulirl. I)og. This figure shows the results obtained on cllromatogrnlIlling tlw II!-of t IllA s~~rum proteins of a female dog. The first spot nhow t h(t origin tlrol~ z:lLw ir :t ghost (11:rt :~ppcars in hydrolyzates of native serum protein I)ut has bren four~~l in our I:ll)or:ttory to be due to amino acids other than the six swn in t h(x s,\,ntlwtic. hy~lrol~~zntcs. They do not appear when thcx six amino acicls are t watr~ll v ith 6 .V II(Il at 100°C. for 24 hr. and chromatogrnmmed as tlescribctl hew. The rc~lntiw intc~rlsititic~s of the glycine and serine (4 and 3) of t,ho cu.%-glol)ulin RW similar to that of ~-globulin. Similarly, the threonine (6) and :rlsnine (6) also put, 1 IIC CY?-in 11w c*I:lss of the p-globulins (see text). The -f-globuliu of t,his run is I,rcsr:ut in slightly less concentration than the rest of the streaks, but the intcnsit> of alanine :III~ threonine are about equal to each other. This distinctly dif’fcww tiates t hr y-globulin from the other proteins where &nine is uniformly mow in tcnsc than thp threoninr.
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can be useful as a survey technique, where subsequent quantitative study may not be possible or desired. All the components considered in Table I have constant proportions of threonine and aspartic acid except for the r-globulins. In the dog, the era-globulins resemble the &globulins. This resemblance indicates that the amino acid character may be indicative of other properties since lipide and hemoglobin in the dog sera migrate with the as-globulins rather than with the ,&globulins as in other species (11). These general observations are based on data which are not independent of large variations of a single amino acid. The data presented in Table I have been recalculated in terms of gram moles of amino acid per 100 moles of aspartic acid and plotted as seen in Fig. 2. Certain general features of the distributions of amino acid in the proteins of the sera of at least three animal species can be deduced from these data. ,,,~p%+q
150
125 100 75
-GLOBULINS
Electrophoretic
-
Component
FIG. 2. The values for gram moles of amino acid per 100 moles aspartic acid are plotted against the major protein groups of dog, rat, and human sera, arranged in order of decreasing mobility.
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1. The Contour of Each Amino Is Similar from one Species to the Next. This indicates a basic uniformity in the pattern of the distribution of these amino acids in the prot’eins of the sera of the dog, rat, and man. Tt shows chiefly that serine and glycine tend to increase nhilc alnninc and glut)amic acid t,end to decrease with decreasing mohilitjy of the elw trophorctic components. This tendewy can also be seen in Fig. 1. 3. The Sum of ihe Two Aliphafic Amino Acids, , 1lanine ontl (;l!ycirre, ire Terms oj Their Molar Ratio per 100 dlol~s ~4sspuwtic -1cd is Relntiwl~~ C’orrstant. In order of decreasing mobility, excluding the y-globulins. in human scra for the four components their sums are: 111, 133, 135, 1-H. 111the dog, including as-globulins, 149, 142, l-43, 13!) and 143. In thch rat, 138, 126, 154, 146. 3. The Reciprocal Relation of Ala+ae to Glyci?rc. &1st,hc ratio of alaninc~ to aspartic falls with decreasing mobility from albumin to /%glohulins, t,he ratio of glycine t.o aspartic acid increases. It, ib as if th(, glyc%~e ww replacing alanine in t,hese proteins. For example (‘I’al)le 11) it, the dog. ill terms of moles amino acid per 100 moles uspartjics, ~L-globulin has about 15 molts more glycinc than albumin, but, “2 moles ICSSalarlille. Again, a?-globulin has 7 moles more glycine than LYE ? but ti moles less nlanine. In the &globulins one finds 10 moles glycine illcwaso and 10 moles alanine decrease. This steady loss of alanine which is rrplawd l)! glycinc wit’11 decreasing rnobility of the protein group e\-c’lt as a first approximatjion from these data appears again in t,he human sera, and is t,ruc of the rat except in the case of Lux-globulins; but, eve11in the rat the> @-globulins cont,ain 40 moles more glycine and 32 moles less nlnnirrc than f he albumin. ;\gain a relationship is seen in all thr components cswpt 1ho ganlmn, in which both the alanine and glycinc arc incrwscd :IR (Y)II~pared t.o the adjacent component the &globulins. k. The (‘onstancy of the Ratio of Glutamic Acid to .4 lnnircc,. ‘1’1~~~lwlar ratio of 111we two amino acids varies betwell 1.3 and 1.5 throughout, thv mtirc range of mobility of the three species with the c>rcqtiolt of the ocz-globulin of t,he rat, which is 1.1 and t,he albumin of the dog whkh is I .G. Of the 16 proteins considered, six values of 1.3, five \-nlucs of I ,4, and three values of 1.5 can be ralculat,ed. 5. The Ratio of Threonine fo Aspartic ,4cid is (‘orcstant \vithin that range of 0.51 to 0.62 for the 16 protein groups considered. 6. Ratio of dlanine to Threonine. In the t,hree allwmins in order of (log, rat, and man, 1.9, I .8, 2.2. cul-Globulin, 2.1, 1..S,,1.4; cu2-globl&l, 1.4. I .4, 1.33; olS-globulin, dog 1.1 ; P-globulins, 1.I, I .I, I .2; y-globnli tw, 0.8, 0.8.
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II
The Value of B (Alanine + Glutamic Acid) and C (Serine + Glycine) Electrophoretic Components of Dog, Rat and Man
of the
The electrophoretic components are arranged in order of the value of n/K to show the relationship of this value to the relative mobility of the components except in the case of the r-globulins. The value of n is the same as moles of unit B, and K is taken as the sum of B and C. These symbols are f om the equation m = (f) [A + nB + (K-n)CI explained in the text. component SOUK‘Z B C *lK Albumin Man 117 -6” 1.00 Albumin Rat 86 6 0.95 Albumin 119 28 0.81 Dog Globulins Alpha-l 56 42 0.57 Dog Alpha-l Man 37 36 0.50 Alpha-2 Man 28 56 0.38 Alpha-l Rat 20 42 0.32 Alpha-2 24 50 0.32 Dog Alpha-2 12 Rat 53 0.18 Beta Rat 2 72 0.03 Beta 2 Man 72 0.03 Alpha-3 0 82 0 Dog 0 Gamma Rat -lb 106 Beta” 18 89 0.16 Dog Gamma Man 20 0.13 138 23 Gamma 138 0.14 Dog a The p-globuhn of the dog is placed closer to the r-globulins 1lecause from these data the CY~ of the dog is probably the true D-globulin and the ,$globulin may be -yr-globulin (see text). b Negative values in these cases are not real but are due to the fact that figures to the closest decade were used in formulating the polypeptide, .i, discussed in the text. Since this polypeptide is made up of the lowest molar ratios of each of the amino acids to aspart,ic acid, values less than zero are not possible. These figures were therefore taken as zero in calculating n/K.
0.8. With but one exception this ratio is indeed one of tile most constant reported here. It places the LYEof the dog with the &glo.bulins. Although it does not differentiate the a1 and albumin in the dog, it does clearly do so in the rat and man. It clearly differentiates the /3- f rorn the r-globulins. 7. The y-Globulins Appear To Be p-Globulins with Excess of Hydroxylated Amino Acids with Some Additional Aliphatics. Thus, in terms of gram moles per 100 moles of aspartic acid, the total moles of serine plus threonine of the r-globulins are found in greater amounts than in the
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p- by the following number of moles in the three species: dog 81; rat 58; man 93. The ratios of the increases of the hydroxy aliphatics to t>he aliphatics is thus close to 5-7 in the dog and man.
The main objectjive of this invest’igation was to establish certain criteria for detecting a change from the normal compositJion of the mixture of proteins normally found in each of t’he Gaussian envelopes of the Tiselius electrophoret’ic pattern. Our approach to this complex problem deals only with the protein moiety of these complex substances, and not their physiological and biological functions, nor t’heir identity as chemical individuals. The amino acid relationships out.lined above can be so treated as to demonstrate certain empirical characteristics of t’he electrophoretic components as prepared here. It is not necessary to determine all of the amino acids of a protein mixture to show that, its composition is different from anot’her group of proteins. An alt’eration in t,he molar ratios of t,wo amino acids may be sufficient,. In the following we have used the molar rat#io of five amino acids to a sisth one. The lowest molar ratio of each amino acid per 100 moles aspartic acid of the 16 mixtures of proteins calculated from Table I are, glmamic acid (90), nlanine (70), threonine (50), serine (40), and glycine (30). Values to the closest decade were t’aken. Thus the artificial empirical compound (Asp,,, , Glut , AlaTo, Threso , Serto , Gly,,) was devised. By subt,racting t’his formulation from the molar ratios of each amino acid per 100 moles aspartic acid of each of the protein mixtures found in Table I, the values in Table II are obtained. From these data the relation of relative mobility, nz, of each component to the relative composition of the mixture of proteins would be described as, m = (f) [A + nB + (K - n)C], where A is represented by aspartic acid and threonine, B by glutamic acid and nlanine, and C by serine and glycine; n is t,he number of moles of glut.amic acid and alanine, and K is the sum of B and C. Table 11 includes values of n,lK to show how this value varies with the average mobility of the component’s. In considering the question whether a given protein is related to the normal serum proteins, the following procedure is proposed in light of the above. (a) The molar ratio of glutamic acid to alanine should be bet’weeri 1.3 and l.(i. (b) The sum of moles of alanine and moles of glycine per 100 moles of aspartic acid should be between 125 and 150.
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Hepatic ,AMINO
ACID + TEMPLATE
f
GLOBULINS
B
I1
B
POOL,
I
* I I I
FIG. 3. This is a diagrammatic representation of a biological implication that might be drawn from these data which would account for the spectrum of mobilities and the shapes and the sizes of the Gaussian envelopes seen in the Tiselius patterns in a variet,y of conditions. Positions on the template refer to those that would be occupied by the synthetic polypeptide, A; a unit characterized by alanine and glutamic acid, B; and a unit characterized by serine and glycine, C, as referred to in the test.
(c) The n/K value and molar ratio of threonine to alanine should be similar to that of the electrophoretic component whose mobility is closest to that of the test protein. By applying these criteria to a number of proteins whose analyses are summarized by Tristram (la), fibrinogen can be shown t’o belong to the family of serum proteins, while the globin of hemoglobin, trypsin, chymotrypsin, p-lactoglobulin do not. To what extent t,he proteins of abnormal sera can be so detected will be reported at a later date. The above scheme applies to the albumins, ol-globulins, and fl-globulins, but not to the r-globulins because of the threonine and serine content. The biological implication of this, which will be subject to future study, has led us to a temporary hypothesis as to the biosynthetic origin of the serum proteins. Since the r-globulins are not formed in the liver and it is recognized that the other globulins can be produced there (13), the serum albumins, and (Y- and fl-globulins, could be formed in a single template as proposed in Fig. 3. Such a scheme can explain the alterations in the protein composition of the Gaussian envelopes found in Tiselius patterns under a variety of conditions (14-17), which today cannot be explained by any one generalization. It does explain how a whole spectrum of proteins of graded mobility can occur as in the case of the serum proteins. The proportions of each component found would depend on the
availability of the amino acids making up the units whicah irwlude flw ones indicated, but do not exclude others. The availahilitjy of alanine and glutamic acid, for example, would determine the extent of albumin formation during metabolic stress. A recent report of Niller and Hale (1% showed that the ratio of albumin and globulin formed by the pwfusctl lircr depended on the level of amino acid intake. This lends support to our concept. The early notions of Kosscl (19i and B1oc.k (20) can 1~. recalled in regard t,o t,he concept of a protein “anlagt” and “orosin” drvclopc~l by these investigators, and Peters (2 I ) and Sorl,hrop (22 1 mow rwcntly, who proposed “int,ermediate” polypcptidcs it1 t.hc pathways of protein biosynthesis.
‘l’ht~ aspartica acid, glutamic acid, glycine, serine, thrcortinc, and alaIlitle contc~~t~sof the cl&rophoretic components of the scrI1m of the clog, rat, and nl:tll ww compared. While glutamica :wid 2nd alalliirc \vt’w found to be charnct’erist,ic of t’he faster components, glycitw a11t1swills w-cre most abundant in the slower component,s n-hctt cnch amino aAt1 was expressed in terms of gram moles of amino wid per 100 moles aspartic acid. The r-globulins of all t)hrce spwies \vcrc distilrguishtd 1)~ {heir high hydroxy amino acid values. (‘ritcria arc proposed for the determination of the presww of a protein nbtrornlal to the serum. These are based OII the relat’iolrship bet wccn t,he molar ratios of cert,ain amino acids and the mobility of the norm:d ,wrum proteins described here.
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11. PITTONI, A., AND TRIVELLATO, E., Atti ist. venels sci., letters ed arts; Classc sci. mat. e nal. 111, 69 (1952-53); cited C. A. 48, 1299Oh (1954). 12. TRISTRAM, G. R., in “The Proteins” (Neurath and Bailey, eds.), Vol. I, part A, p. 181 ff. Academic Press, New York, 1953. 13. MILLER, L. L., BLY, C. G., WATSON, M. I,., AND BALE, W. F., J. Ezpll. Med 94, 431 (1951). 14. WINZLER, R. J., Advances in Cancer Research 1, 503 (1953). 15. ROBERTS, S., AND WHITE, A., J. Riol. Chem. 180,505 (1946). 16. KNAPP, E. I,., AND ROUV~H, J. I,., Pedibics 4, 508 (1949). 17. SCHULTZ, J., SHAY, II., TURTLE, iz., .~NI) GRIJENSTEIN, RI., AlIst. Amer. Chem Sot. 124th Meeting, Chicago, 1!)53,69(i. 18. MILLEH, I,. I,., AND BALE, W. F., k’edercztion I-‘,oc. 11, 260 (1!152), 19. KOSSEL, A. Z. physiol. (Iher,,. 44, 347 (1!)05). 20. BLOCK, R. J., J. Lliol. Chem. 106, 455 (1934). 21. PETERS, T., ./. Biol. Chem. 200, 461 (1953). 22. NORTHROP, J. H., AND PARI’ART, H. K., “Chemistry and Phgsiolog? of Growth.” Princeton Univ. Press, Princeton, 1949.
January
School of Medicine,
3, 1955
INTRODUCTION Zone electrophoresis of animal sera carried out according to the techniques of Kunkel and Slater (1) made possible the preparation of the classical Tiselius electrophoret,ic components of the dog, rat, and man as described in a previous report from this laboratory (2). The possibility that such protein preparations may be identified by means other than mobility was suggested by the data reported by Brand and Edsall (3) on the amino acid composition of some of the proteins of human sera isolated by chemical fractionation procedures. Amino acid paper chromatography of the hydrolyzates of such readily prepared proteins offered an available means for establishing criteria for identification of electrophoretic components that could be used to study changes in protein composition of the major Tiselius groups. This was not possible in terms depending on the quantitative variation in the area of a given range of mobility. Such findings could be applied to any protein mixture that can be separated by zone electrophoresis. In a preliminary report of this study (4, 5), amino acid hydrolyzates of the serum proteins of the rat separated either on paper or starch yielded amino acid chromatograms that showed certain features characteristic of each of the groups of proteins whose mobility fell within major groups of the classical Tiselius pattern. The present report deals with the quantitative aspects of these findings, which have been extended to three animal species, and show that not only are each of the protein fractions of a given species distinct from the other components of the same serum, but that there is a remarkable 174
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SER.4
17.5
similarity between analogous components from one species to the next. This means that the distribution of the amino acids amongst the serum proteins of the normal dog, rat, and man is similar, and that the same amino acids are the characteristic features of a given prot,ein group in t.he three species. The criteria for the identification of the serum proteins are established by considering the electrophoretic pattern to result from a system of proteins whose amino acids constitute a definitive distribution in relation to their electrophoretic mobility. The mobility of any protein, ot)hel than the normal serum proteins found in the serum would not necessarily be related in terms of its own amino acids to the same definitive distribution reported here for the serum proteins. METHODS The methods for the preparation of the protein fractions used in these expcriments have been reported previously (2). In preliminary studies there was reasorlable evidence that glutamic acid, aspartic acid, alanine, glycine, serine and thrconine, which represent two dicarboxylic acids, tFo straight-chain aliphatic acids, and two hydroxglated aliphatic acids, could serve as a good basis for comparing the serum proteins. One decided advantage, besides representing some of the kc,> groups of amino acids, n-as that they could be readily analyzed in a routine fashion as a single streak wherein each amino acid appeared as a single spot. In this R-a: replicate analyses of a single hydrolyzate as recommended by Block et al. (6) were carried out with ease. A given series of streaks were joined together for reading in an automatic densitometer attached to an automatic recorder. Other methods depending on column chromatographic separation were not suitable both from the time required for a given analysis, and the small amount of material available in most cases. For example, after electrophoresis of as little as 0.2-0.4 ml. of serum of a single rat the proteins could be separated and hydrolyzed to yield sufficient material for repeated analysis of amino acids that were suitable to diffrrentiatts the proteins (5).
Hydrolysis H&o~!Js~s and Preparation of Hydrolyzates Jar Amino Acid (‘hrotilatogrclpl1.y. Aliquots of albumin and in most cases the entire globulin fractions were hyclroIyzed in G S HCI for 24 hr. at 100°C. Experiments with trifluoroacetic acigl r+ sulted iI large losses of threonine and serine heretofore not reported l)y others. Treatment of synthetic hydrolyzates of serum albumin 01 r-globulin with trifluoroacetic acid sho~~tl on chromatography distinct losses of these two amino acids. Removal of the IICl with chloroform solutions of di~2~eth~lhexylarnine (Gj did not yield preparations suitable for the chromatographic solvents used in these studies. Direct removal of the acid by evaporation in uacuo, and solution of the residue, iI1 10% isopropyl alcohol proved most satisfactory.
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Chromatography The solvent system used for quantitative amino acid paper chromatography of aspartic acid, glutamic acid, glycine, serine, threonine, and alanine was phenolwater buffered at pH 12, as described by McFarren and Mills (7)~. Acetone added to the butanol-ninhydrin reagent to increase the rate of evaporation of the solvent after color development (8), and precauticns taken to prevent heating the papers in the presence of phenol aided materhlly in obtaining well-developed spots with a minimum of tailing. In some of our experiments the chromatograms were run in ll>s X 435 X 12 in. museum jars in which the paper was suspended by all-glass supports. In most cases, however, a commercial “Chromatocab” was used, which gave more consistent results because >f better control of temperature and humidity. Simultaneous determination of a greater number of samples could be run on a single sheet of paper. In a sample run, the initial concentration of a-amino N was determined on the hydrolyzate by the ninhydrin method proposed by Troll and Cannan (9). From 1 to 10 ~1. of hydrolyzate was placed alternately with standard spots across the bottom of the llaper, so that up to 22 spots which included 11 unknowns were chromatographetl together. From 1 to 4 such analyses were averaged in determining the values fcr a-amino N for each of the amino acids reported here. A number of samples of leaper (Whatman No. 1) were tested by placing 22 spots of the same standard acrok,s the paper, and the density was read after color development. Deviations noted in certain parts of the paper were taken into consideration when adjacent spots due to standard and unknown were found to be “off” the curve which showed the relationship between density versus the logarithm of a-amino N concentratior: . Block et al. (6) suggested that standard hydrolyzates be used which were close in relative amino acid composition to that of the unknown. The standards used in these experiments were therefore made up according to the amino acid composition of human serum albumin, and a-, ,8-, and r-globulin reported by Brand. We have found that only when an analogous component was used as standard in this way, e.g., albumin read against albumin, and globulin against the corresponding globulin, were reproducible results obtained. Another difficulty that was encountered involved the marked variation in concentration of two amino acids such as glycine and glutamic acid of albumin. Here in the lowest concentrations, glutamic acid gives dense spots, while glycine is hardly detectable; as one increases the concentration, the glycine reaches suitable densities only at a point where the glutamic acid spot is too intense to include in the analysis. It is in such cases that the replicate series and repeated runs become necessary.
Calculation of Results The total a-amino N of the six amino acids w-as determined, and the amount of each amino acid was expressed as a percentage of this value for each hydrolyzate analyzed. The terms used are thus mole fraction per cent of the total moles of amino acid represented by the aspartic, glutamic, serine, glycine, threonine, and alanine, of a given protein fraction. In this way errors other than those due to the amino acid determination are reduced to a minimum; for the nintydrin method of Troll and Cannan was found to be useful as a guide only, and the Lowry et al. (10)
Z.(i “I .5 ‘i.5 “4.1 24. I I1 .!I 113..i 1o.:i 1 I .!I 1 I .s I”.0 !I I 11.4 I’.!1 IS.4 I(i.7 IS.2
21.s ‘I :< “2. I ‘I .o ‘L’.(i 1!1.0 l!l.i xi. 1 I 2. :i II :! 1’. I I :i .i Iti. II. I 12. I I’.!1 1:i. 1 Ii.4 I\. I lli. I
Ii 0
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1:i. I
I4 2
l!).!l 21.2 “I .5 21 ..i I!).li 20. I Ii. I I I.!) Il.5 14.e Iti.0 IB. 4 I’.’ 1:io I L’ I I-I .(I II.!) I5.0
ELECTROPHORESIS
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179
acaid values. The variation between species can be seen to be less than that, of the same component within a given species. In Fig. 1, examples of typical chromatograms of the hydrolyzates of the serum proteins of the three species are presented. Certjnin generalities, rwdily drawn from the data of Table I, (WI be observed with th(l naked cyc from these chromatograms. For example, the albumins of wdh species are characterized by the intense ulaninc~ spot when corn pared to the threonine spot. of the same streak; this relation iti not tho santc: in the, streaks of t,he other components. In fact, t’hctr-globulins she\\ more iutnwc thrconine spotIs than alanine, while in t hr CILSUof the @globulins the wversc is seen; t,hus differentiat~ing uot only t hew tjwo :~d,jacw~t~mobility areas, but also the albumin. ‘I’hc glycine-serine spots .W~IX: IO determine the albumin and al-globulins ill the samfb n1au11~~r. .111d nhcll one considers the fluctuations in iutjrnsity, ocz-globulins (WI Iw sew to be different in relative composition from the al- a11d/3-glotw lilts. III o\lr labornt80ry those working with t.hesc c~hromat,ograms h:lr~~ lcwned to guess accurately at the origin of a giver1 streak \vith grwt, rcgularitv. L\lthough we recognize t’hat such obscrvatious arc s~~~)~jwti\~e, w \\-ish to present them as a means of a readily :~vail:~ble clllalitativr~ Iwt of the I1ature of a mixture of proteins from a (wnimoi1 sours that lirr) intlic~ntc the points of application of 3--4 ~1. of synthetic 11ydrolyz:~tc~s corr~w I)nntlirlg to human wrum proteins using the data of Brand and Edsnll (R!. This wl)rwcnt~s :i I otal of 4 pg. of a-amino N that was placed at each origin. Sinw no (131:~ on alaninc~ ~\~re available, this amino acid was eliminatetl from t,hcl h!-clrolJ.z:~t~s, c~src~l)~ for albumin where t,he value for bovine serum albumin \xas usfIll. l’hf> chromatograms were run in buffered phenol at pH 12.0 as descrilwl I)!- 111*F’arR-II :~rr(l Rlills (7) \vith t,hc exception that ascending technique NW use~l. Toil that tlw chnr:rct(lr of each streak is distinct from the other; e.g., t,hc~ low glycirw of nll)umin, thtt high glycine of &globulin and the high serinr of -,-globulirl. I)og. This figure shows the results obtained on cllromatogrnlIlling tlw II!-of t IllA s~~rum proteins of a female dog. The first spot nhow t h(t origin tlrol~ z:lLw ir :t ghost (11:rt :~ppcars in hydrolyzates of native serum protein I)ut has bren four~~l in our I:ll)or:ttory to be due to amino acids other than the six swn in t h(x s,\,ntlwtic. hy~lrol~~zntcs. They do not appear when thcx six amino acicls are t watr~ll v ith 6 .V II(Il at 100°C. for 24 hr. and chromatogrnmmed as tlescribctl hew. The rc~lntiw intc~rlsititic~s of the glycine and serine (4 and 3) of t,ho cu.%-glol)ulin RW similar to that of ~-globulin. Similarly, the threonine (6) and :rlsnine (6) also put, 1 IIC CY?-in 11w c*I:lss of the p-globulins (see text). The -f-globuliu of t,his run is I,rcsr:ut in slightly less concentration than the rest of the streaks, but the intcnsit> of alanine :III~ threonine are about equal to each other. This distinctly dif’fcww tiates t hr y-globulin from the other proteins where &nine is uniformly mow in tcnsc than thp threoninr.
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can be useful as a survey technique, where subsequent quantitative study may not be possible or desired. All the components considered in Table I have constant proportions of threonine and aspartic acid except for the r-globulins. In the dog, the era-globulins resemble the &globulins. This resemblance indicates that the amino acid character may be indicative of other properties since lipide and hemoglobin in the dog sera migrate with the as-globulins rather than with the ,&globulins as in other species (11). These general observations are based on data which are not independent of large variations of a single amino acid. The data presented in Table I have been recalculated in terms of gram moles of amino acid per 100 moles of aspartic acid and plotted as seen in Fig. 2. Certain general features of the distributions of amino acid in the proteins of the sera of at least three animal species can be deduced from these data. ,,,~p%+q
150
125 100 75
-GLOBULINS
Electrophoretic
-
Component
FIG. 2. The values for gram moles of amino acid per 100 moles aspartic acid are plotted against the major protein groups of dog, rat, and human sera, arranged in order of decreasing mobility.
ELECTROPHORESIS
OF ANIiKIL
SER.\
181
1. The Contour of Each Amino Is Similar from one Species to the Next. This indicates a basic uniformity in the pattern of the distribution of these amino acids in the prot’eins of the sera of the dog, rat, and man. Tt shows chiefly that serine and glycine tend to increase nhilc alnninc and glut)amic acid t,end to decrease with decreasing mohilitjy of the elw trophorctic components. This tendewy can also be seen in Fig. 1. 3. The Sum of ihe Two Aliphafic Amino Acids, , 1lanine ontl (;l!ycirre, ire Terms oj Their Molar Ratio per 100 dlol~s ~4sspuwtic -1cd is Relntiwl~~ C’orrstant. In order of decreasing mobility, excluding the y-globulins. in human scra for the four components their sums are: 111, 133, 135, 1-H. 111the dog, including as-globulins, 149, 142, l-43, 13!) and 143. In thch rat, 138, 126, 154, 146. 3. The Reciprocal Relation of Ala+ae to Glyci?rc. &1st,hc ratio of alaninc~ to aspartic falls with decreasing mobility from albumin to /%glohulins, t,he ratio of glycine t.o aspartic acid increases. It, ib as if th(, glyc%~e ww replacing alanine in t,hese proteins. For example (‘I’al)le 11) it, the dog. ill terms of moles amino acid per 100 moles uspartjics, ~L-globulin has about 15 molts more glycinc than albumin, but, “2 moles ICSSalarlille. Again, a?-globulin has 7 moles more glycine than LYE ? but ti moles less nlanine. In the &globulins one finds 10 moles glycine illcwaso and 10 moles alanine decrease. This steady loss of alanine which is rrplawd l)! glycinc wit’11 decreasing rnobility of the protein group e\-c’lt as a first approximatjion from these data appears again in t,he human sera, and is t,ruc of the rat except in the case of Lux-globulins; but, eve11in the rat the> @-globulins cont,ain 40 moles more glycine and 32 moles less nlnnirrc than f he albumin. ;\gain a relationship is seen in all thr components cswpt 1ho ganlmn, in which both the alanine and glycinc arc incrwscd :IR (Y)II~pared t.o the adjacent component the &globulins. k. The (‘onstancy of the Ratio of Glutamic Acid to .4 lnnircc,. ‘1’1~~~lwlar ratio of 111we two amino acids varies betwell 1.3 and 1.5 throughout, thv mtirc range of mobility of the three species with the c>rcqtiolt of the ocz-globulin of t,he rat, which is 1.1 and t,he albumin of the dog whkh is I .G. Of the 16 proteins considered, six values of 1.3, five \-nlucs of I ,4, and three values of 1.5 can be ralculat,ed. 5. The Ratio of Threonine fo Aspartic ,4cid is (‘orcstant \vithin that range of 0.51 to 0.62 for the 16 protein groups considered. 6. Ratio of dlanine to Threonine. In the t,hree allwmins in order of (log, rat, and man, 1.9, I .8, 2.2. cul-Globulin, 2.1, 1..S,,1.4; cu2-globl&l, 1.4. I .4, 1.33; olS-globulin, dog 1.1 ; P-globulins, 1.I, I .I, I .2; y-globnli tw, 0.8, 0.8.
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II
The Value of B (Alanine + Glutamic Acid) and C (Serine + Glycine) Electrophoretic Components of Dog, Rat and Man
of the
The electrophoretic components are arranged in order of the value of n/K to show the relationship of this value to the relative mobility of the components except in the case of the r-globulins. The value of n is the same as moles of unit B, and K is taken as the sum of B and C. These symbols are f om the equation m = (f) [A + nB + (K-n)CI explained in the text. component SOUK‘Z B C *lK Albumin Man 117 -6” 1.00 Albumin Rat 86 6 0.95 Albumin 119 28 0.81 Dog Globulins Alpha-l 56 42 0.57 Dog Alpha-l Man 37 36 0.50 Alpha-2 Man 28 56 0.38 Alpha-l Rat 20 42 0.32 Alpha-2 24 50 0.32 Dog Alpha-2 12 Rat 53 0.18 Beta Rat 2 72 0.03 Beta 2 Man 72 0.03 Alpha-3 0 82 0 Dog 0 Gamma Rat -lb 106 Beta” 18 89 0.16 Dog Gamma Man 20 0.13 138 23 Gamma 138 0.14 Dog a The p-globuhn of the dog is placed closer to the r-globulins 1lecause from these data the CY~ of the dog is probably the true D-globulin and the ,$globulin may be -yr-globulin (see text). b Negative values in these cases are not real but are due to the fact that figures to the closest decade were used in formulating the polypeptide, .i, discussed in the text. Since this polypeptide is made up of the lowest molar ratios of each of the amino acids to aspart,ic acid, values less than zero are not possible. These figures were therefore taken as zero in calculating n/K.
0.8. With but one exception this ratio is indeed one of tile most constant reported here. It places the LYEof the dog with the &glo.bulins. Although it does not differentiate the a1 and albumin in the dog, it does clearly do so in the rat and man. It clearly differentiates the /3- f rorn the r-globulins. 7. The y-Globulins Appear To Be p-Globulins with Excess of Hydroxylated Amino Acids with Some Additional Aliphatics. Thus, in terms of gram moles per 100 moles of aspartic acid, the total moles of serine plus threonine of the r-globulins are found in greater amounts than in the
ELECTROPHORESIS
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183
p- by the following number of moles in the three species: dog 81; rat 58; man 93. The ratios of the increases of the hydroxy aliphatics to t>he aliphatics is thus close to 5-7 in the dog and man.
The main objectjive of this invest’igation was to establish certain criteria for detecting a change from the normal compositJion of the mixture of proteins normally found in each of t’he Gaussian envelopes of the Tiselius electrophoret’ic pattern. Our approach to this complex problem deals only with the protein moiety of these complex substances, and not their physiological and biological functions, nor t’heir identity as chemical individuals. The amino acid relationships out.lined above can be so treated as to demonstrate certain empirical characteristics of t’he electrophoretic components as prepared here. It is not necessary to determine all of the amino acids of a protein mixture to show that, its composition is different from anot’her group of proteins. An alt’eration in t,he molar ratios of t,wo amino acids may be sufficient,. In the following we have used the molar rat#io of five amino acids to a sisth one. The lowest molar ratio of each amino acid per 100 moles aspartic acid of the 16 mixtures of proteins calculated from Table I are, glmamic acid (90), nlanine (70), threonine (50), serine (40), and glycine (30). Values to the closest decade were t’aken. Thus the artificial empirical compound (Asp,,, , Glut , AlaTo, Threso , Serto , Gly,,) was devised. By subt,racting t’his formulation from the molar ratios of each amino acid per 100 moles aspartic acid of each of the protein mixtures found in Table I, the values in Table II are obtained. From these data the relation of relative mobility, nz, of each component to the relative composition of the mixture of proteins would be described as, m = (f) [A + nB + (K - n)C], where A is represented by aspartic acid and threonine, B by glutamic acid and nlanine, and C by serine and glycine; n is t,he number of moles of glut.amic acid and alanine, and K is the sum of B and C. Table 11 includes values of n,lK to show how this value varies with the average mobility of the component’s. In considering the question whether a given protein is related to the normal serum proteins, the following procedure is proposed in light of the above. (a) The molar ratio of glutamic acid to alanine should be bet’weeri 1.3 and l.(i. (b) The sum of moles of alanine and moles of glycine per 100 moles of aspartic acid should be between 125 and 150.
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Hepatic ,AMINO
ACID + TEMPLATE
f
GLOBULINS
B
I1
B
POOL,
I
* I I I
FIG. 3. This is a diagrammatic representation of a biological implication that might be drawn from these data which would account for the spectrum of mobilities and the shapes and the sizes of the Gaussian envelopes seen in the Tiselius patterns in a variet,y of conditions. Positions on the template refer to those that would be occupied by the synthetic polypeptide, A; a unit characterized by alanine and glutamic acid, B; and a unit characterized by serine and glycine, C, as referred to in the test.
(c) The n/K value and molar ratio of threonine to alanine should be similar to that of the electrophoretic component whose mobility is closest to that of the test protein. By applying these criteria to a number of proteins whose analyses are summarized by Tristram (la), fibrinogen can be shown t’o belong to the family of serum proteins, while the globin of hemoglobin, trypsin, chymotrypsin, p-lactoglobulin do not. To what extent t,he proteins of abnormal sera can be so detected will be reported at a later date. The above scheme applies to the albumins, ol-globulins, and fl-globulins, but not to the r-globulins because of the threonine and serine content. The biological implication of this, which will be subject to future study, has led us to a temporary hypothesis as to the biosynthetic origin of the serum proteins. Since the r-globulins are not formed in the liver and it is recognized that the other globulins can be produced there (13), the serum albumins, and (Y- and fl-globulins, could be formed in a single template as proposed in Fig. 3. Such a scheme can explain the alterations in the protein composition of the Gaussian envelopes found in Tiselius patterns under a variety of conditions (14-17), which today cannot be explained by any one generalization. It does explain how a whole spectrum of proteins of graded mobility can occur as in the case of the serum proteins. The proportions of each component found would depend on the
availability of the amino acids making up the units whicah irwlude flw ones indicated, but do not exclude others. The availahilitjy of alanine and glutamic acid, for example, would determine the extent of albumin formation during metabolic stress. A recent report of Niller and Hale (1% showed that the ratio of albumin and globulin formed by the pwfusctl lircr depended on the level of amino acid intake. This lends support to our concept. The early notions of Kosscl (19i and B1oc.k (20) can 1~. recalled in regard t,o t,he concept of a protein “anlagt” and “orosin” drvclopc~l by these investigators, and Peters (2 I ) and Sorl,hrop (22 1 mow rwcntly, who proposed “int,ermediate” polypcptidcs it1 t.hc pathways of protein biosynthesis.
‘l’ht~ aspartica acid, glutamic acid, glycine, serine, thrcortinc, and alaIlitle contc~~t~sof the cl&rophoretic components of the scrI1m of the clog, rat, and nl:tll ww compared. While glutamica :wid 2nd alalliirc \vt’w found to be charnct’erist,ic of t’he faster components, glycitw a11t1swills w-cre most abundant in the slower component,s n-hctt cnch amino aAt1 was expressed in terms of gram moles of amino wid per 100 moles aspartic acid. The r-globulins of all t)hrce spwies \vcrc distilrguishtd 1)~ {heir high hydroxy amino acid values. (‘ritcria arc proposed for the determination of the presww of a protein nbtrornlal to the serum. These are based OII the relat’iolrship bet wccn t,he molar ratios of cert,ain amino acids and the mobility of the norm:d ,wrum proteins described here.
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11. PITTONI, A., AND TRIVELLATO, E., Atti ist. venels sci., letters ed arts; Classc sci. mat. e nal. 111, 69 (1952-53); cited C. A. 48, 1299Oh (1954). 12. TRISTRAM, G. R., in “The Proteins” (Neurath and Bailey, eds.), Vol. I, part A, p. 181 ff. Academic Press, New York, 1953. 13. MILLER, L. L., BLY, C. G., WATSON, M. I,., AND BALE, W. F., J. Ezpll. Med 94, 431 (1951). 14. WINZLER, R. J., Advances in Cancer Research 1, 503 (1953). 15. ROBERTS, S., AND WHITE, A., J. Riol. Chem. 180,505 (1946). 16. KNAPP, E. I,., AND ROUV~H, J. I,., Pedibics 4, 508 (1949). 17. SCHULTZ, J., SHAY, II., TURTLE, iz., .~NI) GRIJENSTEIN, RI., AlIst. Amer. Chem Sot. 124th Meeting, Chicago, 1!)53,69(i. 18. MILLEH, I,. I,., AND BALE, W. F., k’edercztion I-‘,oc. 11, 260 (1!152), 19. KOSSEL, A. Z. physiol. (Iher,,. 44, 347 (1!)05). 20. BLOCK, R. J., J. Lliol. Chem. 106, 455 (1934). 21. PETERS, T., ./. Biol. Chem. 200, 461 (1953). 22. NORTHROP, J. H., AND PARI’ART, H. K., “Chemistry and Phgsiolog? of Growth.” Princeton Univ. Press, Princeton, 1949.