Microheterogeneity of albumin

CLINICA CHIMICA ACTA

116

MICROHETEROGENEITY

J. REJNEK,

T. BEDNARfK

OF

.4ND

ALBUMIN

J. KOtf

Institute of Haematology and Blood Transfusion *, Prague (Czechoslovakia) (Received

March zgth, 1962)

SITMMARY

Very pure preparations of human serum albumin prepared by preparative electrophoresis or ethanol fractionation were subjected to chromatographic analysis on modified cellulose and individual chromatographic fractions were analysed by immunoelectrophoresis or electrophoresis on agar gel. It was demonstrated that albumin can be separated into two distinct fractions, the larger of which can probably be subdivided further. In some chromatographic fractions of the two preparations investigated, the albumin present behaved normally during electrophoresis on agar gel, whereas during immunoelectrophoresis an incomplete precipitation reaction was obtained indicated by the formation of a very weak, diffuse precipitate. It can be assumed that albumin contains molecules with different antigenic structures.

While studying

the problem

of heterogeneous

proteins,

we investigated

recently

the fate of rabbit albumin-% and y-globulin-35S after intraperitoneal administration to ratsIt 2. Even during the first few days after administration, it was possible to detect radioactivity in individual protein fractions of rat serum by electrophoresis on agar gel and subsequent autoradiography. When, however, the same materials were subjected to radioimmunoelectrophoresis, it was found that with the exception of a single precipitation curve (only after administration of albumin) in the cc-globulin area, the precipitates which were formed by the antigen-antibody reaction possessed no radioactivity. These results provided no explanation of the phenomenon, and we concluded that the absence of activity in the precipitates may be due to two causes: either the incorporation of fragments of the heterogeneous labelled protein causes deviations in the structure of the protein molecule which then becomes unable to give the precipitation reaction, or the serum protein contains a certain proportion of molecules which have an incomplete or different antigenic structure. As the first of these alternatives seemed less probable, particularly because several authors provide evidence that protein can be built up only from amino acids or low molecular peptides, we devoted our attention to the second alternative. The heterogeneity of proteins and particularly of albumin has been investigated and discussed by many authors but has not been directly proved3. We therefore attempted to fractionate very pure * Director:

Prof. J. Ho&EJ~~, M.D.,

D.Sc. C/in. Chim. Acta, 8 (1963) 116-126

Fig. r. Electrophoresis on agar gel and immunoelectrophoresis of albumin prepared by ethanol fractionation. On strips I, 3 and 5 normal human serum; on strips z and 4 albumin.

Fig. a. Electrophoresis on agar gel and immunoelectrophoresis of fractions obtained by separation of normal serum by preparative electrophoresis on agar gel. On strip I initial serum; on strips s-8 individual fractions. On strip 3 analysis of albumin which was used for the experiments.

preparations of albumin by chromatography on modified cellulose and to characterise the resulting fractions immunochemically.

MATERIAL AND METHODS

Hwnan serum albumin @.wified by ethanol fractionation was prepared by the method described previously4. The fraction obtained (after separating fractions I, II + III and IV by COHN’S procedures) which contained g5-g6% albumin, 2-30/b cc-globulin and I-2% P-globulins was dissolved in water to reduce the ethanol concentration to 10%. The PH remained unaltered, i.e. 4.8 & 0.1. To this slightly turbid solution 12.5 vol.% of cation exchange resin and 25 vol. o/0of anion exchanger were added in the H+ and OH- forms respectively and the mixture was agitated for 15 min. Clin. Chint. .4&z, 8 (1963)

116-126

J. REJNEK et Ul.

11s

The ion-exchange resins were subsequently removed by filtration on a silon mesh and the solution was cleared by filtration through a layer of asbestos and cellulose and a layer of silica. The solution thus obtained was diluted with an equal volume of cold pyrogen-free, distilled water and subjected to lyophilization. The prepared albumin contains a very low percentage of salts (it complies with the requirements of salt-poor albumin), it does not contain thermolabile globulins and its terminal stability is better than in classical preparations. The electrophoretic and immuno-electrophoretic examination of the prepared albumin is shown in Fig. I. Human serum albumin isolated electrophoretically was prepared from normal pooled sera from blood donors by preparative electrophoresis in agar gel*. The results of electrophoresis on agar gel and immunoelectrophoresis of the preparation are shown in Fig. 2. For further experiments the albumin fraction from strip no. 3 was used. Immunoelectrophoresis and electrophoresis on agar gel were carried out by the micromethod described previously7. The precipitation antihuman serum used was a commercial preparation of the Pasteur Institute, lot 511. Chromatography on DEAE cellulose. of albumin on a column of DEAE cellulose which prepared by resin in buffer A and by sedimentation of the suspension in a column 20 x I cm. The specimens were applied to the column in the usual manner; 5 ml of a 3% solution was always used. The solutions of both albumins were dialyzed before separation at o” for r6-r8h against buffer A. During the gradient elution of the column the buffers used were those described for a similar purpose by PETERSON et a1.8. 2.5 g of ion-exchange

A = 0.005 AZ H3P04 + 0.04 M TRIS (hydroxymethylaminomethane) R = 0.35 M HSPO, + 0.35 M TRIS

PH

=

8.6

PH

=

4.0

The rate of flow through the column was 6 ml/h, and the fractions were collected for 3o-min periods. In order to facilitate comparison of the elution diagrams of both albumins, all experiments were made on the same column which was regenerated by means of 0.01 AT 1\TaOH.

Identification of precipitating proteivzs was carried out by HIRSCHFELD’S method 9 under the same conditions as for the immunoelectrophoresis quoted above. The upper channel (for the precipitation serum) in the vicinity of the analysed sample after electrophoretic separation was normally filled with precipitation serum and the lower channel first with a solution of immunochemically homogeneous albumin for a period of 40 min and then with the precipitation serum. After the precipitation was complete, only those components of the lower part of the analysis which did not correspond to albumin had precipitated while in the upper part of the analysis all components precipitated. RESULTS 3%

solutions

3 and 4. As is apparent

of albumin, prepared by ethanol fractionation in agar gel were subjected to chromatographic The results of both experiments are given in Figs.

of both specimens

or separated by electrophoresis separation on modified cellulose.

from Fig. 3 the extinction

values at 280 m,u at the very beginning Clin. Chim. Acta, 8 (1963) I 16-120

MICROHETEROGENEITY

of the elution

diagram

obtained

OF ALBUMIN

by chromatography

of albumin-ethanol,

119 form two

consecutive maxima; subsequently, the values decline and reach the main maximum only after a relatively long interval. The ascending part of this maximum rises very steeply compared with the descending part which by its asymmetry indicates the presence of yet another component. After this peak no further maximum was detected. Somewhat different is the picture obtained from chromatography of albumin

NUMBER OF FRACTION

Fig. 3, Elution diagram of ethanol-albumin.

NUMEER OF FRAC TION

Fig. 4. Elution diagram of albumin prepared by electrophoresis.

prepared by electrophoresis (Fig. 4). At the beginning there are again two maxima, the first of which, in contrast to Fig. 3, is very low, much lower than the second one. The main peak is in this case symmetrical and the presence of another component is suggested only by very slight waves at the bottom of the descending part of the main peak. In subsequent work we carried out electrophoretic and immunochemical control of all fractions obtained by chromatography, in order to evaluate whether all maxima are due to protein and if so, whether they correspond actually to albumin or to some ballast protein present in the original material. It is not possible to show diagrammaClin. Chim. Acta, 8 (1963) 116-126

120

J. REJNEK et Ul.

tically all the analyses carried out, hence we shall describe only those which represent individual peaks on the elution diagrams. In Fig. 5 are shown the electrophoresis (on agar gel) and immunoelectrophoresis of fractions corresponding to both the initial maxima of Fig. 3. In ethanol-albumin,

Fig. 5. Electrophoresis on agar gel and immunoelectrophoresis of fractions corresponding to the two initial maxima of Fig. 3 (ethanol-albumin). On strip I normal serum; on strips 2-5 analyses of fractions s-5 (first peak) ; on strip 6 analysis of fraction 6 (second peak).

Fig. 6. Immunochemical identification of fractions of first maximum of ethanol-albumin. On strip I normal serum: on strips 2-6 fractions corresponding to equivalently marked ones on fig. 3.

in the first maximum, zones with albumin mobility were found (fractions 2, 3, 4, 5) while in the second one no protein was detected. In the case of the albumin which was obtained electrophoretically, no protein was detected in any of the investigated fractions. Although the zones obtained on the two types of electropherograms of the first maximum of ethanol-albumin corresponded on the whole in their mobility, they differed in shape from the usual zone of serum albumin. Therefore we tried to identify them using HIRSCHFELD’S method (Fig. 6). During this analysis immunoelectrophoretically homogeneous albumin was used for saturation (its analysis is given on strip 2 (‘Zin. C-him. Acta, 8 (1963) 116-126

MICROHETEROGENEITY

OF ALBUMIN

121

of Fig. z), hence the results of this experiment can be taken as evidence that the controversial zones represent albumin. During investigations of subsequent fractions it was found that protein could be detected only in fractions where a further peak appeared on the elution diagram. Fig. 7 shows electrophoretic and immunoelectrophoretic analyses of fractions cor-

Fig. 7. Electrophoresis on agar gel and immunoelectrophoresis of fractions corresponding to the main peaks of the elution diagrams of the two investigated specimens of albumin. On strips 1-6 analysis of fractions Nos. 31,33,35,38,41 and 45 (corresponding to equally marked ones on Fig. 3) ; on strips 8-13 analysis of fractions 23, 26, 28, 30, 31 and 32 (corresponding to equally marked ones on Fig. 4) ; on strips 7 and r4 analyses of normal sera.

responding to the main peak of the elution diagram of both investigated albumins. In both instances, throughout the area of the main maximum, zones can be detected whose mobility corresponds to that of albumin. As is apparent from Fig. 7, individual zones on agar-electrophoresis, particularly as regards anaIyses of fractions of ethanolalbumin, differ considerably in shape. Therefore we also identified the individual Clin.Clzim.Acta,8 (1963) 1x6-126

122

J. REJNEK

et d.

fractions in those specimens (Fig. 8). It was found that all zones corresponded to serum albumin. Analyses by immunoelectrophoresis and electrophoresis on agar-gel of further fractions are given in Fig. g. In all samples, the fractions prepared from ethanol-albumin and analyzed by electrophoresis on agar gel again show zones in the albumin area which, as in the case of electrophoretically prepared albumin, are much less intense than in samples shown in previous figures. This is because these fractions are final and the ratio of albumin is relatively small. Despite this, the albumin zones are visible in all specimens and are of approximately equal intensity, except for the specimens given on the first strips. A quite different picture is, however,

Fig. 8. Immunochemical identification of fractions: in the left half of the figure is ethanol-albumin, where fractions 31. 33, 35, 38, 41 and 45 of the elution diagram correspond to strips 1-6 of Fig. 3 ; on the right half are albumin fractions prepared by electrophoreris. To strips 1-6 correspond fractions 23, 26, 28, 30, 31 and 32 of the elution diagram in Fig. 4. On strips No. 7 analysis of normal serum.

found in the immunoelectrophoretic analyses. In fractions obtained from ethanolalbumin we can see that the first two samples precipitate normally, i.e. they give the normal distinct precipitation line. Subsequent samples, shown on strips 4-6, form only very weak precipitation lines which are not easily visible and their precipitation reaction is apparently incomplete, though the zones found by electrophoresis on agar gel do not differ substantially from the first two. Similar, though not quite so marked, results were obtained during immunoelectrophoretic analysis of albumin fractions obtained by electrophoretic separation. While on the first and second strip there is a marked precipitation zone, on the third strip it is less distinct and on the remaining three there are already indistinct lines, as was found for ethanol-albumin. This phenomenon of different immunochemical reactivity could lead to the assumption that the fractions shown in Fig. 9 do not involve merely albumin but also some cr-globulin fraction. Therefore we also analysed these specimens by HIRSCHFELD’S method (Fig. IO). -4s in the above-mentioned cases, it was found that albumin was present in all specimens investigated.

MICROHETEROGENEITY OF ALBUMIN

I23

DISCUSSION The problem

of heterogeneity

by HUGHES lo who, however, the majority

of crystalline

relied mainly

of which can, according

plasma

albumin

on the results

has been discussed

of electrophoretic

to FOSTER 3, be accounted

studies,

for by isomerization.

Fig. 9. Electrophoresis on agar gel and immunoelectrophoresis of further fractions. On strips 1-6, fractions 47, 50, 56, 57, 58, 59 from ethanol-albumin; on strips S-13 fractions 33, 34, 35, 36, 37 and 38 from albumin isolated by electrophoresis. Strips 7 and 14 illustrate analysis of normal serum. Better evidence of heterogeneity methods. human

was obtained

recently by extended

chromatographic

SWINGLE AND TISSELIUS~~, TISSELIUS et ~1.~~ and HJERT~~N~~subjected serum albumin

able to separate

to chromatographic

it into 2-3

components.

analysis Similar

and BOMAN AND WESTLUND~~, who obtained

2-4

PETERSON AND SOBER’~ these results may possibly

on calcium

phosphate

results were obtained peaks on Dowex-2.

According

be due to dimerization C/is

Chim.

Ada,

and were

by BOMAN~* to

of protein

8 (1963)

116-126

124

J. REJNEK et at.

or its reaction with smaller molecules. SOBER et a1.l’ were able to separate human serum albumin into three components by chromatography on DEAE cellulose. Similarly, KELLER AND BLACK~~ analyzed chromatographically beef mercaptoalbumin and human serum albumin and showed that one of the three components of human albumin differs from the remaining two in its cysteine content. Electrophoretic investigations of the homogeneity of albumin were made recently by RESSLER'~, who found that by Smithies’ technique human serum albumin can be separated into several components; he identified the components and proved the absence of globulin fractions by immunoelectrophoresis. Similarly, SAIFER et aZ. 2o used electrophoresis on starch gel for investigations of purified albumin. They found that several components were obtained only from ethanol-albumin; albumin ob-

Fig. IO. Immunochemical identification of fractions: in the left half of ethanol-albumin, where to strips 1-6 correspond fractions 47, 50, 56, diagram in Fig. 3 ; on the right half are albumin fractions prepared by 1-6 correspond fractions 33, 34, 35, 36, 37, 38 of the elution diagram in of normal serum.

the figure are fractions of 57, 58, 59 of the elution electrophoresis. To strips Fig. 4. On strip 7 analysis

tained by preparative electrophoresis on paper gave only a single component. The above authors assume therefore that the albumin sub-fractions may be formed during the preparation and purification of albumin. In addition to this more or less convincing evidence of the heterogeneity of albumin, we may also quote the clear indirect proof, i.e. that serum albumin contains less than one sulfhydryl group per molecule and that thus albumin must exist which contains these groups as well as albumin which does not3, 21. Our results submitted in this paper also confirm the heterogeneity of albumin fractions. Two distinct maxima, the first of which at the start of the elution diagram is particularly marked in ethanol-albumin (Fig. 3), in addition to the asymmetry of the descending part of the main maximum, provide evidence that the investigated albumin contains several fractions which can be separated by the method used. The finding that in albumin which is obtained electrophoretically the first maximum is very low and the asymmetry of the descending part is indicated only by waves at the

MICROHETEROGENEITY OF ALBUMIN

12.5

base of the main maximum, is obviously due to the fact that in this case (see Fig. z), only a part of the serum albumin fraction separated by electrophoresis on agar gel was investigated. It might be possible that the initial maxima in both elution diagrams are due to the presence of protein impurities, particularly a-globulin, in both the preparations investigated (Figs. I and 2). However, immunoelectrophoresis, electrophoresis on agar gel, and particularly HIRSCHFELD’S analyses, showed that in ethanol-albumin the fractions which account for the above-mentioned maximum always contain albumin which immunochemically is identical with serum albumin even if the shape of its precipitation zone, like the zones on the agar-electropherogram, is somewhat unusual. In the first maximum-in albumin prepared by electrophoresis-it proved impossible to detect protein, perhaps because its concentration was lower than the range of sensitivity of the methods used. It is also of interest to study more closely the immunochemical and electrophoretie analyses of the chromatographic fractions which form the main maximum. In the ascending part of the main maximum of the elution diagram of ethanol-albumin (Fig. 7; Nos. 31, 33, 35), normally migrating and precipitating albumin is present; in addition there is ccl-globulin in fractions 31 and 33 which is apparent only on immunoelectrophoresis and which was also detected in the initial material. In the descending part (Fig. 7; Nos. 38, 41) there is also normally precipitating albumin but its zones on electrophoresis on agar gel differ markedly in shape not only from the zones of the ascending part but also from each other. In fraction 45 albumin is again found but in a considerably lower concentration. HIRSCHFELD’S analyses provide evidence that it is albumin in all instances. This difference in shape of the zones on the agarelectropherogram confirms that asymmetry of the descending part of the elution diagram is actually due to the presence of several different albumin fractions. As far as albumin obtained by electrophoresis is concerned, among the fractions which form the main maximum of the elution diagram (Fig. 7; Nos. 23, 26, 28, 30, 31, 32), fraction No. 23 contains impure cr,-globulin which gave a normal picture in all analyses which were carried out. That no differences were observed here in individual fractions on the electropherogram on agar gel can be explained by the fact that only part of the serum albumin fraction was used as starting material. In preparative electrophoresis on agar gel probably a partial fractionation of albumin occurs; although there is no visible separation of the albumin zone, in view of the microheterogeneity we may assume a different composition of the frontal and cathodic portions of this zone. Immunoelectrophoresis and electrophoresis on agar gel of chromatographic fractions corresponding to the remaining part of the elution diagram of ethanolalbumin indicate the probable answer to the question set in the introduction of this paper, i.e. whether albumin may contain some molecules which during immunoelectrophoresis do not give a precipitation reaction with antiserum though the antiserum reacts in a satisfactory way with the complete albumin fractions. As is apparent from Fig. g, fractions 47, 50, 56, 57, 58 and 59 form zones on the electropherogram on agar gel, which by their mobility and shape correspond to albumin and are, except for NO. 47, of approximately equal size and intensity. On the other hand, the immunoelectropherogram indicates that the normal precipitation reaction is given only by fractions 47 and 50, while the others give a weak diffuse precipitate which correClin. Chim.

Acta, 8

(1963) 116-126

J. REJNEK Pt Ul.

126

sponds to the normal precipitate in shape but not at all in intensity. The appearance of the precipitate suggests that some, though not all, of the determinant groups which are essential for a normal antigen-antibody reaction are lacking so that the precipitation reaction was incomplete. A similar phenomenon can be observed in the last chromatographic fractions of albumin prepared by electrophoresis (Fig. 9). Although the zones on the agar-electropherogram (fractions 33-38) are of approximately equal size and intensity, on the immunoelectropherogram only fractions 33 and 34 precipitate properly; fraction 35 precipitates less distinctly and fractions 36, 37 and 38 precipitate very diffusely. This behaviour is similar to the previous case. To summarize all the results obtained, all the analytical methods applied helped to confirm the microheterogeneity of albumin. Chromatography on DEAE cellulocc gave a separation into two distinct separate fractions (in both instances the presence of albumin was proved by HIRSCHFELD’S reaction). The larger one of the two fractions could be separated into further fractions, as was confirmed by the different shapes of the albumin zones obtained by electrophoresis on agar gel. Immunoelectrophoresis of the final fractions of the eluate after chromatography of ethanol-albumin as well as electrophoretic albumin, showed the existence of albumin which gi\-es \vith normal commercial antihuman serum an incomplete precipitation reaction; a weak, diffuse precipitate is formed. The investigation supports our hypothesis, which was made during the study of resynthesis after the administration of biosyntheticall> labelled albumin, that a part of the albumin molecule exhibits a different antigenic structure from the remainder of the molecule. RIIFERFXCBS 1 J, REJXEK, T. BEDNAR~K 2 T. REDNAR~K, 1. REJNEK

AND AND

V. KNESSLOVA, \T. KXESSLOVA,

Physiol.

Bohemoslove~~.,

Pilysiol.Bohemoslown..

in in

the press. the mess.