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Isolation and characterization of albumin from porcine serum, colostrum and urine
Int. .I. Biochem.,
1977, Vol. 8, pp. 285 CO 294. Pergamon
Press. Printed
in Great Britain
ISOLATION AND CHARACTERIZATION OF ALBUMIN FROM PORCINE SERUM, COLOSTRUM AND URINE ROLAND N. K. CARLSSON,BENGT I. INGVARSSONAND B6w~ W. KARLSSON Institute of Zoophysidlogy, University of Lund, S-223 62 Lund, Sweden (Received
22 October 1976)
Abstract-l. Albumins from adult swine serum, foetal serum, colostrum and urine from neonatal pigs were isolated by the use of immunosorbent techniques. 2. The purity of the isolated albumins was controlled by polyacrylamide gel-electrophoresis, electroimmunoassay and by immunization of rabbits with isolated albumin. 3. Gel-filtration showed that the mol. wts of adult serum albumin, foetal serum glbumin and colostrum albumin were 67,500, 66,500 and 65,000, respectively. Urine albumin was shown to be composed of 2 populations with mol. wts of 65,500 and 44,500. Determination of the sedimentation coefficients confirm$d these results. 4. Albumin from neonatal pig urine has an amino acid composition intermediary to that of colostrum and foetal serum, while a similar composition was found for colostrum and adult serum albumin. 5. Isoelectric focusing showed a similar PI-pattern for adult and foetal serum albumin on the one hand and urine and colostrum albumin on the other hand while neonatal serum albumin showed an intermediate pattern. 6. The data obtained confirmed previous findings that albumin in colostrum originates from sow’s serum. After onset of colostrum ingestion colostrum albumin is transported via the intestinal absorptive cells to the blood of the neonatal pig and is then excreted into the urine during the period of transient proteinuria.
INTRODUCTION Many different methods have been employed for the isolation of albumin as reviewed by Janatova (1974) and by Peters (1975). The most common isolation procedures include precipitation with inorganic agents (Cohn et al., 1940a; Spencer & King, 1971) or with organic agents (Cohn et al., 1940b; Michael, 1962; Iwata et al., 1968; Gambal, 1971; Jimenez et al., 1974). The advantage of these methods is that they yield large quantities of fairly pure albumin. Other methods include anionic exchange chromatography on DEAE-cellulose (Spencer & King, 1971; Keller & Block, 1959), gel-filtration (Ribarac-StepiC & Kanazir, 1967) and preparative electrophoresis (Jungblut & Turba, 1963). More recently afiity chromatography has been used. These methods utilize the specific affinity between albumin and ligands immobilized on a matrix. The ligands used include fatty acids (Peters et al., 1973), bilirubin (Hierowski & Brodersen, 1974) and blue dextran (Travis & Pannell, 1973). After the discovery by Luetscher (1939) of multiple bands in the electrophoresis pattern of human serum albumin near pH 4, much interest has been focused on the presence of and the reasons for the heterogeneity of albumin. These investigations have been mainly concerned with the heterogeneity of albumin in serum and have been reviewed by Janatova (1974). From earlier studies it is evident that foetal, neonatal and adult pig serum, sow’s colostrum and milk, and urine of neonatal piglets during the period of transient proteinuria all contain similar forms of albumin (Karlsson, 1970; Bergelin & Karlsson, 1974; Bergelin & Karlsson, 1975). Questions have arisen as to whether the albumins detected in these various B.C.8/4--c
body fluids of the pig are identical as regards molecular structure and physiological function. In order to attempt to answer these questions we isolated albumin from swine serum, foetal serum, sow’s colostrum and neonatal swine urine. The aim of the study was to compare these albumins with regard to mol. wt, sedimentation coefficient, isoelectric point, amino acid composition and antigenic determinants. For this reason it was necessary to find a gentle isolation procedure which yielded a pure preparation of albumin. W e t h ere fore employed a technique involving affinity chromatography on immunosorbent columns. We were encouraged to use this method from our previous experiments on the isolation of alpha-foetoprotein from foetal pig serum (Carlsson et al., 1976). MATERIAL AND METHODS Experimental
animals and
collection
of
samples
Serum from pig foetuses and adult pigs was obtained at the slaughter of sows at Scanek, KBvlinge, as described earlier (Carlsson et al., 1976). A total of 100 pig foetuses with crown to rump lengths (C-R length) of 13-22 cm were used. Urine and serum from neonatal piglets, aged lo-24 hr. were collected at Svenska HushNningssdlskapets i MalmGhus Iln FarsGksgHrd, BjHrsjBlagird, as described by Bergelin and Karlsson (1974). Urine contaminated with faeces or blood was excluded. The urine samples used for analyses were kept at +4”C overnight, centrifuged at 3000 9 and frozen at -20°C until further use. Urine used for gel-filtration and isoelectric focusing was concentrated by ultrafiltration through an UM 10 diaflo membrane in an Amicon cell. Colostrum was collected by hand-milking sows during farrowing. Colostrum was skimmed by centrifugation at +4”C at 20,000 9 for 60min in a LKB 65 B centrifuge. The samples were frozen until further use. 285
286 Immune
ROLAND
N. K. CARLSSON, BENCT I. INGVARSSONAND B~~RJEW. KARLSSON
sera
Antisera to the albumin fractions isolated from foetal and adult serum, colostrum and neonatal urine were produced in rabbits. Each rabbit was given 5 ml of antigen solution emulsified in 5 ml of Freunds incomplete adjuvant (Difco, Detroit). Eighty % of the antigemadjuvant emulsion was administered under the scapula, while 20% was administered intramuscularly in one hind leg. The animals were injected twice, with a 6-week interval. Two rabbits were used for each albumin preparation. Using this procedure a total of 6.0mg of swine serum albumin @S-albumin), foetal serum albumin (FS-albumin) and colostrum albumin (Col-albumin) and 4.0mg of neonatal urine albumin was injected. One week after the second injection blood was collected by heart puncture and the antisera were obtained. Antisera against foetal and adult serum and colostrum were prepared as described elsewhere (Karlsson, 1970). Isolation procedures The procedures for isolating the various albumins are shown in Table 1. At the first stage (a-e in Table 1) serum albumin from adult pigs was isolated, monospecific antibodies were produced and coupled to Sepharose. At the second stage (f-h in Table 1) this immunosorbent column was utilized for the isolation of albumin from adult serum (SS), foetal serum (FS), colostrum (Col) and neonatal urine. The initial isolation of albumin from adult pig serum included 2 subsequent runs of preparative electrophoresis in pevicone C-870 (Kema Nord) as described earlier (Carlsson et al., 1976). Briefly, 7 ml swine serum was applied and electrophoresis was run for 40 hr. The part containing albumin was isolated and subjected to a second preparative electrophoresis in order to further eliminate contaminating proteins. This procedure rendered a pool of crude albumin (b in Table 1). Three ml of this pool, containing approx 60 mg albumin, was applied to a Con-A Sepharose column (Pharmacia Chemicals) previously equilibrated Table
1. Procedure
of isolation
of SS-albumin,
with 0.1 M acetate buffer, pH 6.0 containing 1 M NaCl, 10-3M CaCl,, 10w3M MgClz and 10m3M MnCl,. The eluate was monitored using a Uvicord II LKB Beckman spectrophotometer. After the first peak had been collected, a 2% alpha-D-methylglucoside solution in acetate buffer, pH 6.0, was added to the column and a second peak containing the glucoproteins was obtained. The first peak from the Con-A Sepharose contained all the albumin applied and was concentrated by negative pressure dialysis to a final volume of 3 ml (c in Table 1). A sufficient volume of this solution was added to an immunosorbent column prepared with antiserum to adult swine serum (ASS) so that the capacity of the column for albumin was exceeded but the saturation limit for the contaminating proteins was not exceeded. This ASS-immunosorbent column was prepared according to Porath et al. (1973) as described earlier (Carlsson et al., 1976). The amount of albumin which exceeded the capacity of the column was eluted with a 0.075 M Tris-HCl buffer containing 0.075 M NaCl and lOA M CaCIZ, pH 8.1 (d in Table 1). The proteins adsorbed by the column, e.g. contaminating proteins and albumin, were eluted using a buffer containing 0.075 M Tris-HCl, 0.15 M NaCl, 0.2 M tetramethylendiamine and 10m3 M CaCl,, pH 11, (Dr. Ulla-Britt Hansson, Institute of Biochemistry, Lund, to be published). The protein content was measured at 280 nm using a Uvicord II LKB Beckman spectrophotometer. Antiserum to the isolated SS-albumin was produced and designated ASSA (e in Table 1). The y-globulin fraction from this monospecific antiserum was isolated by precipitation with saturated ammonium sulphate as described earlier (Carlsson et al., 1976). An ASSA-immunosorbent column was manufactured according to Porath et al. (1973). CNBr activated Sepharose 4B (60g) was allowed to react with 500 mg of the y-fraction dissolved in borate buffer, pH 9.0. Eighty-five % of the protein was bound to the activated Sepharose. The washed product was packed in a column 1.6 x 40 cm FS-albumin,
Col-albumin
and urine albumin
ss (a)
.... .. . . .. . .. ... .. _.. . .
I I
(b)
..
. . . . . . . . . . . . . . . . . . . Crude
Preparative
electrophoresis
Preparative
electrophoresis
SS-albumin Affinity
(c)
. . .. .
. .
chromatography
with Con-A
Sepharose
.. .. I Immunosorbance
I
with ASS-Sepharose
(d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pure SS-albumin Immunization
of rabbits
I (e) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monospecific antiserum (ASSA) Coupling
to sepharose SS, FS, Col, Urine
(f)
I . . . . . . . . . . . . . .._.__............ ASSA Sepharose
(g)
.
...
.
.‘. .
.. ..
.. .
. . I. .
.. ..
I
ASS Sepharose (h)
.
.
. .. .
. Pure
I SS-albumin
Pure FS-albumin Pure Col-albumin Pure urine albumin
281
Albumins from porcine serum, colostrum and urine and equilibrated with the elution buffer (0.075 M Tris-HCl, 0.5 M NaCl. 10W3M CaCI,. uH 8.1) before use. Albumin from swine ser-u& foe&l serum, sow’s colosbum, and neonatal pig urine was isolated in a two-step procedure (f-h in Table 1) involving affinity chromatography first on the ASSA-immunosorbent column and then on the ASS-immunosorbent column. In order to utilize the capacity of the ASSA-immunosorbent column for albumin, different volumes were added, e.g. 0.15 ml SS, 0.3ml Co1 diluted with 2ml Tris-HCl buffer, pH 8.1, 4 ml FS and 15 ml urine all containing about 4 mg alb~in. The pH 11 peak (g in Table 1) was immediately neutralized with 1 M HCl: and concentrated to a tinal volume of 2 ml. A pool of 10 ml of each preparation of albumin containing 20mg albumin was subjected to the ASS-immunosorbent column, utilizing the same principles as described above. The isolated albumins in the pH 8.1 peak (h in Table 1) were concentrated, and showed to contain about Smg of albumin. ~lectpophores~s, imm~~assay and determination of proteipl content
Electrophoresis in 1% agarose gel (Ca-Verona1 buffer, pH 8.6) was run according to Johansson (1972) for the electrophoretic identification of proteins. ~munoelec~ophoresis was performed in order to determine the potency and specificity of the different antisera and to control the purity and electrophoretic mobility of the albumin preparations. Electroimmunoassay was performed according to Laurell (1966) in order to quantitate the different albumins using the isolated pure SS-albumin as a standard. This method was also used to detect minute amounts of antigens contaminating the albumin preparations. Crossed i~unoelectrophoresis was performed on cover glass plates (5 x 5 cm) in agarose gel containing antibodies principally as described by Ganrot (1972). This method was used as a complement for the detection of contaminating antigens and for comparison between the different albumins as regards heterogeneity. Determination of the protein content was carried out according to Lowry eh al. (1951) with bovine serum albumin fraction V (Sigma) as standard. Gel-filtration In order to determine the mol. wts of the albumins, gel-filtration was performed at +4”C on a 2.5 x 80cm Sephadex G-200 column buffered with a 0.05 M Tri+HCl buffer and 0.15 M NaCl. nH 7.5. as described earlier (Carlsson ef ai, 1976). The me&od of Andrews (1965) was used. Soybean trypsin inhibitor (Sigma) mol. wt 21,500; ovalbumin (Grade-V, Sigma) mol. wt 45,000; bovine albumin (fraction V. Sigma) mol. wt 67,000 and 134,000 and anorfer;itin (Serva, Hiidelberg) mol. & 480,000 were used & reference proteins. One millilitre swine serum, 0.75 ml colostrum diluted with 2 ml elution buffer, 3 ml foetal serum and 30 ml urine ultra-coated to 3 ml were applied. The ~stribution of albumin was determined using ele~roimmunoassay and crossed immunoelectrophoresis. Hectrophoresis
in polyucrylamide
gels
Polyacrylamide gel-electrophoresis was performed on an Ortec annaratus in 0.065 M Tris-borate buffer., PH 9.0. __ using a gradiporegel 426% as described earlier (Car&son et al., 1976). L
Ultra-centrijkgation
The sedimentation coefficient was measured in a Spinco Model E Ultracentrifuge at 59,780 rev/min, 259,700 g as described earlier (Carlsson et al., 1976). The sedimentation coefficient s&,_ was calculated by extrapolating to zero protein concentration.
Amino acid composition
Amino acid analysis was performed with a Jeol-6AH automatic amino acid analyzer as described previously (Carlsson et al., 19’76). Isoelectric focusing Isoelectric focusing was carried out on a 110 ml LKB column for 90 hr with a voltage from 450-1000 V, at a pH range from 4 to 6 built up by Ampholine Carrier ampholytes (LKB). A sucrose density gradient was used as a stabilizing medium. The samples were collected in 2.5 ml fractions using an Ultrorac 7000 fraction collector. The pH was measured on a Bergman and Beving pH meter at the same temperature as that used during isoelectric focusing (+ 3°C). Eight milligrams of SS-albumin, FS-albumin. Col-albumin and neonatal serum albumin obtained after preparative electrophoresis and 8 mg urine albumin obtained from 30 ml neonatal urine concentrated by ultrafiltration to 2 ml were focused. Focusing was also performed on 8 mg SS-albumin and 8 mg Co&albumin pretreated with charcoal (Grave) at nH 3, &d at +4”C for 1 hr according to Chen (1967). The albumin distribution in the pH gradient was determined by means of electroimmunoassay. RESULTS adult urine
Isolation
of ul~u~~~ from
colostrurn
and neonatal
serum,
foetal
serum,
Despite, two electrophoretic runs on pevicone, the fractions of SS-containing albumin were contaminated with three other proteins. After a%inity chromatography on Con-A Sepharose which has the ability to bind g~ucoprote~s (page, 1973), two of these proteins were removed. By means of the ASS-immuno-
sorbent column, as described above, the last contaminating protein was removed and pure SS-albumin was obtained (Table 1 a-d and Fig. 1). When the purity of the albumin preparation was assayed by electro~unoassay, ~munoelectrophoresis and crossed immunoelectrophoresis with ASS as antiserum, only one precipitation arc could be seen (Fig. 1). In addition, electrophoresis in polyacrylamide gels showed one distinct band and immunization of rabbits with the isolated albumin resulted in a monospecific antiserum (Table le). Although the antiserum used for production of the ASSA-immunosorbent column was monospec~c when tested by immunoelectrophoresis it was not possible to obtain pure albumin after chromatography on this column. The preparations were found to be contaminated by minute amounts of one protein with the same electrophoretic mobility as that of albumin. This conta~nating protein was present in the prep~ations of albumin from SS, FS, colostrum and neonatal urine (Fig. l), However, it was possible to remove it by means of affinity chromatography on the ASS-immunosorbent column as described above. The albumins thus obtained were proved to be pure by the production of monospecific antibodies after immunization of rabbits. Moreover. in tests with electroi~~oassay, crossed immunoelectrophbresis and immunoelectrophoresis using antisera to adult serum, foetal serum and colostrum, SS-, FS- and CM-albumin gave one precipitation arc (Figs. 1 and 2). However, the albumin from urine showed a different pattern as was best seen by crossed immunoel~trophoresis where two peaks showing
ROLAND N.
288
K.
CARLSON, BENGT I. INGVARSON AND B~RJE W. KARLSON
Fig. 1. Elcctroimmunoassay showing the purification steps for adult swine strum albumin (A, B), foetal serum albumin (C), colostrum albumin (D) and urine albumin (E). Part 4 shows swine serum (a) and fractions obtained after preparative electrophoresis (b). Con-A affinity chromatography (c) and ASS immunosorbance (d). Parts BE show whole serum (c) and fractions obtained after ASSA immunosorhance (f) and fractions obtained after ASS immunosorbance (g). An antiserum to adult swine strum was used (0.03 ml/ml agarosc).
partial identity wcrc formed (Fig. 2D). These results were confirmed by clcctrophoresis in polyacrylamide gels. In addition to the one distinct band revealed by SS-, FS-, Col- and urine albumin the latter showed
IDF--lr----!
Fig. 2. Crossed immunoclcctrophorcsis on isolated albumins from adult swine serum (A), foetal serum (B), colostrum (C), neonatal urine (D) and neonatal urine (E) after addition of equal amounts of isolated albumin from swine serum, foctal strum or colostrum. In (F) the neonatal albumin protile of whole neonatal serum is shown. In gels A E the same picture was ohtaincd using antisera to adult swine strum. foctal serum, colostrum or adult serum alhumin. In gel F an antiserum to adult serum albumin was used. The dark spots in the lower left corner of the gels indicate sample application sites.
Table
2. Molecular
s&,,
Mol. wf und sedimentation corjficient The mol. wts and sedimentation coefficients (s&).~.) of the albumins arc listed in Table 2. From this table it can be seen that the differences in mol. wts found by gcl-filtration wcrc confirmed by the dctcrminations of the sedimentation coefficients. Urine albumin consists of two components with different mol. wts and sedimentation coellicients. When the fractions obtained after gel-filtration of the urine were tested by crossed immunoclcctrophorcsis it was seen that the larger
weight and sedimentation coefficient (s!, ,) of SS-albllmin albumin, Col-albumin and urine album&
SS-albumin Mol. wt
a second band (Fig. 3). This hctcrogcncity. further investigated by crossed immunoclcctrophorcsis. was found in both concentrated otherwise untreated urine and in the pure preparation of urine albumin. The proportion between the two types of albumin found varied between different samples. Addition of SS-, FSor Col-albumin to urine albumin resulted in enlargement of the slow moving albumin fraction while the fast moving fraction was unaffected (Fig. 2E). When serum from piglets aged 24-hr was tested no albumin heterogeneity could be observed (Fig. 2F). Addition of SS, I-S, Co1 or urine to the antisera against the various albumins yielded complete absorption as revealed by immunoclcctrophorcsis. In contrast to earlier findings (Carlsson er al., 1976) no Con-A reactivity of H-albumin could be seen.
FS-albumin
Col-albumin
urine albumin
67,500
66,500
65,000
4.58 + 0.05
4.55 * 0.08
4.52 + 0.04
a Due to small amounts
this value could
not be measured
65,500 44,500 4.46 & 0.19 3.1”
more exactly
W-
289
ide gradipors gel 4-26% run for 160 minutes. showing from Hi 1, . 1 . . . --_-..
291
Albumins from porcine serum, colostrum and urine
(b)
0 40
5.0
6.0
pH-values 4. Distribution of albumin after isoelectric focusing in the pa-range 4-6. Figure (a) demonstrates adult swine serum albumin (O-O), foetal serum albumin (A-A) and neonatal serum albumin (04). Figure (b) shows colostrum albumin (O---U) and neonatal urine albumin (A-A) while Fig. (c) shows charcoal-treated swine serum albumin (t-0) and charcoal-treated colostrum albumin (W---B). component (mol. wt 65,500) corresponded to the slow migrating fraction (first peak), while the smaller component (mol. wt 44,500) corresponded to the fast migrating fraction (second peak) (Fig. 2D).
Isoelectric focusing. The albumin distribution in a pH gradient from 4 to 6 after isoelectric focusing is shown in Fig. 4. SS-albumin and FS-albumin follow the same overall distribution pattern with major peak values around pH 5.5 (Fig. 4a), while colostrum and urine albumin have major peak values at pH 4.5 (Fig. 4b). The albumin from neonatal serum has two large peaks, one at pH 4.5 the other at pH 5.5 (Fig. 4a). It is notable that all the albumins except urine albumin have peak values around the same pH, e.g. 4.5, 4.9 and 5.5, but that the proportion between the peaks differs for the various albumins. Treatment of SSalbumin and Col-albumin with charcoal did not change the SS-albumin distribution significantly whereas the major peak of Col-albumin changed from pH 4.5 to 4.9 (Fig. 4~). Amino acid composition. The amino acid composition of the albumins is shown in Table 3. Almost identical values were found for Col-albumin and SSalbumin with minor variations in glutamine and histidine content. The amino acid composition of FSalbumin differs from that of SS-albumin and Colalbumin, major differences being seen in serine, proline, leucine and lysine content and minor differences in asparagine, valine, cysteine, tyrosine and phenylalanine content. The composition of urine albumin shows intermediate values between E&albumin and Col-albumin on the one hand and FS-albumin on the other for 9 amino acids, while 5 amino acids showed higher values and 3 lower values.
DISCUSSION The principle used for the isolation of swine serum albumin (SS-albumin) described in this paper involves the preparation of a crude fraction of albumin and subsequent chromatography on an immunosorbent column to which antibodies against swine serum (ASS) was coupled. Though tedious, this method can
Table 3. Amino acid composition of SS-albumin, Col-albumin, FS-albumin and urine albumin
Asparagine Tbreonine Serine Glutamine Proline Glycine Alanine l/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan
SS-albumin
Col-albumin
FS-albumin
urine albumin
84 43 38 169 55 29 83 58 59 8 34 93 33 50 100 20 45 not measured
84 44 39 159 55 29 82 61 62 6 36 93 31 49 95 30 43 not measured
75 39 50 170 90 35 16 50 51 5 31 79 42 58 85 24 39 not measured
94 41 41 163 63 44 78 54 58 10 34 84 29 46 86 31 30 not measured
The values are expressed as moles/1000 moles of amino acids.
292
ROLAND N. K. CARLSSON.BENGT I. INGVARSSONAND B~RJE W. KARLSSON
be valuable not only for the isolation of albumin but also other proteins provided that it is possible to obtain a crude preparation in which the protein in question is predominant. However, it is important to be aware of possible sources of error which may occur when the albumins from adult and foetal swine serum, colostrum and urine are isolated using antibodies against adult swine serum albumin in the ASSA-immunosorbent step. This method of preparation could cause a loss in specific albumin components as FS-, Col- and urine albumin may contain specific parts not common to the SS-albumin molecule. However, this risk must be considered to be merely theoretical as such a molecule must lack antigenic determinants present in the SS-albumin molecules. Moreover, the absorption studies, as revealed by immunoelectrophoresis, showed that the different isolated albumins were immunologically identical as the antibodies produced were common to the various albumins. The fact that chromatography on the ASSAimmunosorbent column did not render pure albumin seems remarkable as the antiserum (ASSA) used for preparation of the immunosorbent column proved to be monospecific by the immunological test methods used. It could depend on the presence of minute amounts of undetectable contaminating antibodies, or on binding of the contaminating protein to the fixed anti-albumin-antibodies or on unspecific binding of the contaminating protein to the Sepharose matrix. The results from the investigation of the physicochemical properties of the albumins indicate that differences do exist. These differences may be due to the environment in which albumins are found, or to the developmental stage of the animal or may reflect functional differences. The great similarities in amino acid composition between SS-albumin and Col-albumin indicate that Col-albumin is derived from the serum of the lactating sow. This is in accordance with earlier findings (Polis et al., 1950; Laurel1 & Morgan, 1965; Morgan, 1968; Jennes, 1974). An explanation of the differences noted between SS- and Col-albumin concerning mol. wt, sedimentation coefficient and isoelectric-focusing pattern must be sought in variations in conformation and bound substances such as fatty acids and bilirubin. The results from isoelectric focusing of charcoaltreated Col-albumin and SS-albumin support the assumption that there are differences in the fatty acids bound to Col-albumin and SS-albumin as the p1 pattern of charcoal-treated Col-albumin is strongly affected compared to that of native Col-albumin, whereas charcoal-treated SS-albumin is not significantly affected. The results on charcoal-treated Colalbumin are in agreement with those of Valmet (1969) and Rosseneu-Motreff et al. (1970) who found an increase in p1 after treatment of human serum albumin and bovine serum albumin with charcoal. However, Valmet states that fatty acid-albumin complexes are unstable below pH 5 and that the fatty acids are separated from the albumin molecule during the electrofocusing procedure. This could offer an explanation to the fact that charcoal-treatment of SSalbumin in our study did not affect the p1 as the focusing was run for 90 hr with a voltage of 1000 V during the last 60 hr, with a consequent separation
of the fatty acid-albumin complexes. As the same procedure was used for electro-focusing of Colalbumin we cannot conclude at the present stage that the p1 peak at 4.5 of Col-albumin is caused by the interaction of fatty acids, although treatment with charcoal affected the p1 in a way typical for defatted albumins. The amino acid composition of FS-albumin differs from that of SS-albumin and Col-albumin. This observation supports the findings of Miyoshi et al. (1966) who found a foetal type of human albumin (Alb F) different from that of the adult (Alb A). However, the differences in amino acid composition found in our study do not result in any changes in the p1 pattern of FS-albumin as compared to SS-albumin. This may depend on the fact that the major variations in amino acid composition occurred in non-polar and uncharged amino acids. Moreover it can be concluded that the variations in amino acid composition do not give rise to any immunogenic determinants not common to FS and SS as complete absorption was obtained both when anti-FS-albumin was absorbed with SS-albumin and vice versa. Bergelin and Karlsson (1975) stated that urine albumin from neonatal piglets has a mol. wt of 50,000. This value is based on tests with gel-filtration and subsequent electroimmunoassay. From the present work it is obvious that urine albumin is heterogeneous and consists of at least two components with mol. wts of 65,500 and 44,500. In order to visualize this heterogeneity, electroimmunoassay is an inappropriate test method and crossed immunoelectrophoresis must be used. It has been known for many years that intact proteins are absorbed via the gut of the neonatal pig (Nordbring & Olsson, 1957; Munn & Smith, 1974). Though Page (1969) assumed that the neonatal synthesis of albumin could be totally responsible for the observed abrupt rise in the concentration of albumin, Karlsson (1970) is of the opinion that absorption from colostrum is at least partly responsible for this elevation in concentration. The fact that it was possible to detect the major p1 peaks of both Col-albumin and FS-albumin in the p1 pattern of serum albumin from neonatal piglets aged 24 hr is interpreted to be due to the existence of albumin of both foetal and colostral origin in the neonatal piglet. Results from our investigation on neonatal urine albumin suggest that this albumin partly originates from colostrum. The p1 pattern is very similar to that of Col-albumin with one large peak at pH 4.5. Furthermore, both colostrum albumin and the large component of urine albumin have almost identical mol. wts (65,000 and 65,500) and the precipitates formed during crossed immunoelectrophoresis show a tailing towards the application very common for colostrum albumin, neonatal serum albumin and neonatal urine albumin. The larger component of neonatal urine albumin (mol. wt 65,500) is probably identical to that found in the serum of the neonatal piglet, since addition of SS-, FS- or Col-albumin to the samples or urine albumin as revealed by crossed immunoelectrophoresis increased the amount of the slow migrating fraction without the formation of new peaks. Moreover, the determination of mol. wt, sedimentation coeffi-
Albumins from porcine serum, colostrum and urine cient and the results from polyacrylamide gel-electrophoresis lend further support to this conclusion, The smaller component of neonatal urine albumin (mol. wt 44,500) cannot be detected in serum from piglets aged 24 hr. It seems likely that this component is a breakdown product of the larger one. Although our results indicate that the breakdown may occur in the kidney or in the urine, it cannot be excluded that the breakdown takes place in the intestine before or during the absorption of proteins. ~artinsson (1973) found that urine ~bu~n from neonatal piglets aged 12 hr consisted of 2 fragments and that addition of pure swine albumin revealed a new peak as seen in crossed immunoelectrophoresis. Our finding that intact albumin occurs in the urine can be explained by the fact that we used a pool of urine from piglets aged 10-24 hr. The results From ammo acid analysis on total urine albumin indicate that the albumins found in urine may originate from the foetus as well as from colostrum, as values intermediate to those of Col- and FSalbumin were found for 9 amino acids. For another 8 ammo acids values which were either higher or lower than the corresponding values for Col-albumin and FS-alb~in were found. A possible expl~ation for this deviation may be that the small mol. wt albumin component in neonatal urine is selected during the isolation procedure and has an amino acid composition different from that of the high mol. wt albumin component. The results from this investigation suggest that colostrum albumin originates from sow’s serum. After onset of colostrum ingestion this albumin is absorbed via the intestinal absorptive cells into the bloodstream of the newborn piglet. Therefore the albumin in the serum and urine of the newborn piglet after onset of colostrum ingestion is a mixture of albumin from colostrum and albumin synthesized by the piglet itself. We feel that research in the field on the various albumin types in the body fluids of the pig could give results which would be of use in the investigation of the function of, for example, the intestinal mucosa cells and the cells in the nephron in the kidney of the newborn tion.
piglet after the onset of colostrum
inges-
Acknowledgements-The skilful technical assistance of Mrs. Marie Adler-Maihofer and the secretarial help of Miss Marianne Andersson are gratefully acknowledged. We are also indebted to Dr. Ulla-Britt Hansson at the Biochemical Institute for performing the sedimentation analysis, and to Miss Ing-Britt Johansson at the University Hospital in Lund for performing the amino acid analysis. This investigation was supported by the Royal Physiographical Society, Lund. REFERENCES
ANDREWS P. (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606. BERGELINI. S. S. & KAR~~SONB. W. (1974) Colostrum, serum and kidney proteins in the urine of the developing pig. Int. J. Biochem. 5, 885-894. BWGELINI. S. S. & KARLSSONB. W. (1975) Proteinuria in neonatal piglets after oral and intravenous administration of homologous colostrum and serum proteins. int. J. Biochem. 6, 821-827.
293
CARLSSON R. N. K., INGVARSSON B. I. & KARLSSONB. W. (1976) Isolation and characterization of alpha-foetoprotein from foetal pigs. Int. J. Biochem. 7, 13-20. CHEN R. F, (1967) Removal of fatty acids from serum albumin by charcoal treatment. f. b&I. Chem 242, 173-181. COHN E. J., MCMEEKINT. L., ONCLEYJ. L., NEWELLJ. M. & HUGE?.W. L. (194Ou) Preparation and properties of serum: and plasma proteins-I. Size and charge of proteins separating upon equilibration across membranes with ammonium sulfate solutions of controlled pH, ionic strength and temperature. J. Am. &em. Sot. 62, 33863393. COHN E. J., LUETSCHER J. A., ONCLEVJ. L., ARMSTRONG S. H. & DAVISB. D. (194Ob)Preparation and properties of serum and plasma proteins--HI. Size and charge of proteins separating upon equilibration across membranes with ethanol-water mixtures of controlled pH, ionic strength and temperature. J. Ana. &em. Sot. 62, 33963400.GAMBALD. (1971) Simple, rapid procedure for isolating serum albumin. Biochim. biophvs. Acta 251, 54-56. GANR~T P. 0. (1972) Crossed- immunoelectrophoresis. Stand. J. c2in. Lab. Invest. 29, Suppl. 124, 39-41. H~OWSKI M. & BRODERSEN R. (1974) Covalent binding of bilirubin to agarose and use of the product for &nity chromatography of serum albumin. B~uc~~~. b~u~bys. Acta 354, 121-129. IVVATAT.. IWATAH. & HOLLAM) J. F. (1968) Isolation of albumin from human serum by means of trichloroacetic acid and ethanol. Clin. Chem. Acta 14, 22-30. JANATOV~ J. (1974) On the heterogeneity of serum albumin. J. Med. 5, 149--216. JENNESR. (1974) Biosynthesis and composition of milk. J. invest. ~e~rnato~.63, 109-118. J~ENEZ M. I., CARBALLOB., AYUSO-PARRILMM. S. & PARRILLAR. (1974) A procedure for the rapid isolation of serum albumin using a combination of polyethyleneglycol and ethanol. Biochim. biophys. Acta 372, 436-439. JOHANSSON B. G. (1972) Agarose gel electrophoresis. Stand. J. clin. Lab. &est. iS,&ppl.-124, 7-19: JUNCZBLUT P. W. & TLTRE~A F. 119631Veraleich von Darstel1ungsVe~~ren fti Ratten-~ern~~b~in und Charakterisierung eines reinen Praparates. Biochem. Z. 337, E-103. KARLSSONB. W. (1970) Fetoprotein and albumin levels in the blood serum of developing neonatal pigs. Comp. Biochem. Physiol. 34, 535-546. KELLERS. & BLOCKR. J. (1959) Ammo acid composition of the serum proteins-III. The chromatographic isolation of human and bovine serum albumins, and the amino composition of the fractions. Archs Biuchem. Biouhvs. 85, 3666373. L.&ELL C.-B. & MORGAN E. H. (1965) The relation between the proteins of plasma and milk in the rat. Biochinz. biophyi. Acta loo,-128-135. LACXELLC.-B. (1966) Quantitative estimation of proteins by electrophoresis in agarose gel cont~ning antibodies. Analyt. Biochem. 15, 4552. LOWRY0. H., R~~EBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. LUETSCHER J. A. (1939) Serum albumin-II. Identification of more than one albumin in horse and human serum by electrophoretic mobility in acid solution. J. Am. &em. Sot. 61, 2888-2890. MARTINSSON K. (1973) Immunological identification of the protein components in urine of newborn piglets with relation to intestinal protein transmission. Zbl. Vet. Med. B. 20, 639-647. MICHAELS. E. (1962) The isolation of albumin from blood serum or plasma by means of organic solvents. Biochem. ‘7. 82. 212-218.
294
ROLANDN. K. Cn~.~s.so~?,BENGT I. INGVARSSON AND B~~RJEW. KARLWN
MIYOS~IK., SAIJOK., KOTANIY., KASHIWAG~ T. & KAWAI H. (1966) Characteristic properties of fetal human albumin (Alb F) in isomerization equilibrium. Tokusfaima J. exp. Med. 13, 121-128. MORGAN E. H. (1968) Plasma protein turnover and transmission to the milk in the rat. 13iochim. biophys. Acta 154, 478487. MUNN E. A. & S1111lr-r M. W. (1974) Uptake of albumin by neonatal pig ileum incubated in vitro. J. Physiol., Land. 242. 30-32. NORDBR~NGF. & OLSSON B. (1957)Electrophoretic and immunolo~~ai studies on sera of young pigs. Acta Sot. med. Uppsa~u 62, 193-212. PAGE L. A. (1969) In viuo and in vitro studies on the synthesis of albumin by perinatal pigs. Develop. BioE. 20, 1255145. PAGE?M. (1973) a-Foetoprotein: purification on sepharoselinked Concanavalin-A. Can. J. Biochem. 51, 1213-1215. PETERS T., TANIUCHII-I. & ANFI~SENC. B. (1973) Affinity chromatography of serum albumin with fatty acids immobilized on agarose. J. bial. Chem. 248, 244772451. PETERS T. (1975) Serum albumin. In The Plasma Proteins (Edited by PUTNAMF. W.) Vol. 1, pp, 133-181. Academic Press, London.
B. D., SHMUKLER H. W. & CUSTERJ. H. (1950) Isolation of a crystalline albumin from milk. _I. biol. Chem. 187, 349-354. PORATHJ., ASPBERGK., DREVINH. & Ax& R. (1973) Preparation of cyanogen bromide-activated agarose gels. I. Pam
Chromatog. 86, 53-56.
RIRARAC-STEPI~ N. & KANAUR D. (1967) A method for isolation and purification of hen’s serum albumin. Arch biol. Sci. 19, 5-10. ROSSENEU-MOTREFF M. Y., BLATONV., DECLERCQB. & PEETERSH. (1970) The contribution of bound fatty acids to the heterogeneity of BSA. In Protides of the ~~o~o~ca~ Fluids (Edited by PETERS H.) Vol. 18, pp. 503-508. Pergamon Press. Oxford. SPENCERE. M. & KING T. P. (1971) Isoelectric heterogeneity of bovine plasma albumin. .I. biol. Gem. 246, 201-208. TRAVISJ. & PANNELLR. (1973) Selective removal of albumin from plasma by affinity ~hromato~aphy. C&n. chim. Acta 49, 4952.
VALMETE. (1969) The heterogeneity of human serum albumin. In Protides of the Biological Fhids (Edited by PEETERSH.) Vol. 17, pp. 443-448. Pergamon Press, Oxford.
1977, Vol. 8, pp. 285 CO 294. Pergamon
Press. Printed
in Great Britain
ISOLATION AND CHARACTERIZATION OF ALBUMIN FROM PORCINE SERUM, COLOSTRUM AND URINE ROLAND N. K. CARLSSON,BENGT I. INGVARSSONAND B6w~ W. KARLSSON Institute of Zoophysidlogy, University of Lund, S-223 62 Lund, Sweden (Received
22 October 1976)
Abstract-l. Albumins from adult swine serum, foetal serum, colostrum and urine from neonatal pigs were isolated by the use of immunosorbent techniques. 2. The purity of the isolated albumins was controlled by polyacrylamide gel-electrophoresis, electroimmunoassay and by immunization of rabbits with isolated albumin. 3. Gel-filtration showed that the mol. wts of adult serum albumin, foetal serum glbumin and colostrum albumin were 67,500, 66,500 and 65,000, respectively. Urine albumin was shown to be composed of 2 populations with mol. wts of 65,500 and 44,500. Determination of the sedimentation coefficients confirm$d these results. 4. Albumin from neonatal pig urine has an amino acid composition intermediary to that of colostrum and foetal serum, while a similar composition was found for colostrum and adult serum albumin. 5. Isoelectric focusing showed a similar PI-pattern for adult and foetal serum albumin on the one hand and urine and colostrum albumin on the other hand while neonatal serum albumin showed an intermediate pattern. 6. The data obtained confirmed previous findings that albumin in colostrum originates from sow’s serum. After onset of colostrum ingestion colostrum albumin is transported via the intestinal absorptive cells to the blood of the neonatal pig and is then excreted into the urine during the period of transient proteinuria.
INTRODUCTION Many different methods have been employed for the isolation of albumin as reviewed by Janatova (1974) and by Peters (1975). The most common isolation procedures include precipitation with inorganic agents (Cohn et al., 1940a; Spencer & King, 1971) or with organic agents (Cohn et al., 1940b; Michael, 1962; Iwata et al., 1968; Gambal, 1971; Jimenez et al., 1974). The advantage of these methods is that they yield large quantities of fairly pure albumin. Other methods include anionic exchange chromatography on DEAE-cellulose (Spencer & King, 1971; Keller & Block, 1959), gel-filtration (Ribarac-StepiC & Kanazir, 1967) and preparative electrophoresis (Jungblut & Turba, 1963). More recently afiity chromatography has been used. These methods utilize the specific affinity between albumin and ligands immobilized on a matrix. The ligands used include fatty acids (Peters et al., 1973), bilirubin (Hierowski & Brodersen, 1974) and blue dextran (Travis & Pannell, 1973). After the discovery by Luetscher (1939) of multiple bands in the electrophoresis pattern of human serum albumin near pH 4, much interest has been focused on the presence of and the reasons for the heterogeneity of albumin. These investigations have been mainly concerned with the heterogeneity of albumin in serum and have been reviewed by Janatova (1974). From earlier studies it is evident that foetal, neonatal and adult pig serum, sow’s colostrum and milk, and urine of neonatal piglets during the period of transient proteinuria all contain similar forms of albumin (Karlsson, 1970; Bergelin & Karlsson, 1974; Bergelin & Karlsson, 1975). Questions have arisen as to whether the albumins detected in these various B.C.8/4--c
body fluids of the pig are identical as regards molecular structure and physiological function. In order to attempt to answer these questions we isolated albumin from swine serum, foetal serum, sow’s colostrum and neonatal swine urine. The aim of the study was to compare these albumins with regard to mol. wt, sedimentation coefficient, isoelectric point, amino acid composition and antigenic determinants. For this reason it was necessary to find a gentle isolation procedure which yielded a pure preparation of albumin. W e t h ere fore employed a technique involving affinity chromatography on immunosorbent columns. We were encouraged to use this method from our previous experiments on the isolation of alpha-foetoprotein from foetal pig serum (Carlsson et al., 1976). MATERIAL AND METHODS Experimental
animals and
collection
of
samples
Serum from pig foetuses and adult pigs was obtained at the slaughter of sows at Scanek, KBvlinge, as described earlier (Carlsson et al., 1976). A total of 100 pig foetuses with crown to rump lengths (C-R length) of 13-22 cm were used. Urine and serum from neonatal piglets, aged lo-24 hr. were collected at Svenska HushNningssdlskapets i MalmGhus Iln FarsGksgHrd, BjHrsjBlagird, as described by Bergelin and Karlsson (1974). Urine contaminated with faeces or blood was excluded. The urine samples used for analyses were kept at +4”C overnight, centrifuged at 3000 9 and frozen at -20°C until further use. Urine used for gel-filtration and isoelectric focusing was concentrated by ultrafiltration through an UM 10 diaflo membrane in an Amicon cell. Colostrum was collected by hand-milking sows during farrowing. Colostrum was skimmed by centrifugation at +4”C at 20,000 9 for 60min in a LKB 65 B centrifuge. The samples were frozen until further use. 285
286 Immune
ROLAND
N. K. CARLSSON, BENCT I. INGVARSSONAND B~~RJEW. KARLSSON
sera
Antisera to the albumin fractions isolated from foetal and adult serum, colostrum and neonatal urine were produced in rabbits. Each rabbit was given 5 ml of antigen solution emulsified in 5 ml of Freunds incomplete adjuvant (Difco, Detroit). Eighty % of the antigemadjuvant emulsion was administered under the scapula, while 20% was administered intramuscularly in one hind leg. The animals were injected twice, with a 6-week interval. Two rabbits were used for each albumin preparation. Using this procedure a total of 6.0mg of swine serum albumin @S-albumin), foetal serum albumin (FS-albumin) and colostrum albumin (Col-albumin) and 4.0mg of neonatal urine albumin was injected. One week after the second injection blood was collected by heart puncture and the antisera were obtained. Antisera against foetal and adult serum and colostrum were prepared as described elsewhere (Karlsson, 1970). Isolation procedures The procedures for isolating the various albumins are shown in Table 1. At the first stage (a-e in Table 1) serum albumin from adult pigs was isolated, monospecific antibodies were produced and coupled to Sepharose. At the second stage (f-h in Table 1) this immunosorbent column was utilized for the isolation of albumin from adult serum (SS), foetal serum (FS), colostrum (Col) and neonatal urine. The initial isolation of albumin from adult pig serum included 2 subsequent runs of preparative electrophoresis in pevicone C-870 (Kema Nord) as described earlier (Carlsson et al., 1976). Briefly, 7 ml swine serum was applied and electrophoresis was run for 40 hr. The part containing albumin was isolated and subjected to a second preparative electrophoresis in order to further eliminate contaminating proteins. This procedure rendered a pool of crude albumin (b in Table 1). Three ml of this pool, containing approx 60 mg albumin, was applied to a Con-A Sepharose column (Pharmacia Chemicals) previously equilibrated Table
1. Procedure
of isolation
of SS-albumin,
with 0.1 M acetate buffer, pH 6.0 containing 1 M NaCl, 10-3M CaCl,, 10w3M MgClz and 10m3M MnCl,. The eluate was monitored using a Uvicord II LKB Beckman spectrophotometer. After the first peak had been collected, a 2% alpha-D-methylglucoside solution in acetate buffer, pH 6.0, was added to the column and a second peak containing the glucoproteins was obtained. The first peak from the Con-A Sepharose contained all the albumin applied and was concentrated by negative pressure dialysis to a final volume of 3 ml (c in Table 1). A sufficient volume of this solution was added to an immunosorbent column prepared with antiserum to adult swine serum (ASS) so that the capacity of the column for albumin was exceeded but the saturation limit for the contaminating proteins was not exceeded. This ASS-immunosorbent column was prepared according to Porath et al. (1973) as described earlier (Carlsson et al., 1976). The amount of albumin which exceeded the capacity of the column was eluted with a 0.075 M Tris-HCl buffer containing 0.075 M NaCl and lOA M CaCIZ, pH 8.1 (d in Table 1). The proteins adsorbed by the column, e.g. contaminating proteins and albumin, were eluted using a buffer containing 0.075 M Tris-HCl, 0.15 M NaCl, 0.2 M tetramethylendiamine and 10m3 M CaCl,, pH 11, (Dr. Ulla-Britt Hansson, Institute of Biochemistry, Lund, to be published). The protein content was measured at 280 nm using a Uvicord II LKB Beckman spectrophotometer. Antiserum to the isolated SS-albumin was produced and designated ASSA (e in Table 1). The y-globulin fraction from this monospecific antiserum was isolated by precipitation with saturated ammonium sulphate as described earlier (Carlsson et al., 1976). An ASSA-immunosorbent column was manufactured according to Porath et al. (1973). CNBr activated Sepharose 4B (60g) was allowed to react with 500 mg of the y-fraction dissolved in borate buffer, pH 9.0. Eighty-five % of the protein was bound to the activated Sepharose. The washed product was packed in a column 1.6 x 40 cm FS-albumin,
Col-albumin
and urine albumin
ss (a)
.... .. . . .. . .. ... .. _.. . .
I I
(b)
..
. . . . . . . . . . . . . . . . . . . Crude
Preparative
electrophoresis
Preparative
electrophoresis
SS-albumin Affinity
(c)
. . .. .
. .
chromatography
with Con-A
Sepharose
.. .. I Immunosorbance
I
with ASS-Sepharose
(d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pure SS-albumin Immunization
of rabbits
I (e) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monospecific antiserum (ASSA) Coupling
to sepharose SS, FS, Col, Urine
(f)
I . . . . . . . . . . . . . .._.__............ ASSA Sepharose
(g)
.
...
.
.‘. .
.. ..
.. .
. . I. .
.. ..
I
ASS Sepharose (h)
.
.
. .. .
. Pure
I SS-albumin
Pure FS-albumin Pure Col-albumin Pure urine albumin
281
Albumins from porcine serum, colostrum and urine and equilibrated with the elution buffer (0.075 M Tris-HCl, 0.5 M NaCl. 10W3M CaCI,. uH 8.1) before use. Albumin from swine ser-u& foe&l serum, sow’s colosbum, and neonatal pig urine was isolated in a two-step procedure (f-h in Table 1) involving affinity chromatography first on the ASSA-immunosorbent column and then on the ASS-immunosorbent column. In order to utilize the capacity of the ASSA-immunosorbent column for albumin, different volumes were added, e.g. 0.15 ml SS, 0.3ml Co1 diluted with 2ml Tris-HCl buffer, pH 8.1, 4 ml FS and 15 ml urine all containing about 4 mg alb~in. The pH 11 peak (g in Table 1) was immediately neutralized with 1 M HCl: and concentrated to a tinal volume of 2 ml. A pool of 10 ml of each preparation of albumin containing 20mg albumin was subjected to the ASS-immunosorbent column, utilizing the same principles as described above. The isolated albumins in the pH 8.1 peak (h in Table 1) were concentrated, and showed to contain about Smg of albumin. ~lectpophores~s, imm~~assay and determination of proteipl content
Electrophoresis in 1% agarose gel (Ca-Verona1 buffer, pH 8.6) was run according to Johansson (1972) for the electrophoretic identification of proteins. ~munoelec~ophoresis was performed in order to determine the potency and specificity of the different antisera and to control the purity and electrophoretic mobility of the albumin preparations. Electroimmunoassay was performed according to Laurell (1966) in order to quantitate the different albumins using the isolated pure SS-albumin as a standard. This method was also used to detect minute amounts of antigens contaminating the albumin preparations. Crossed i~unoelectrophoresis was performed on cover glass plates (5 x 5 cm) in agarose gel containing antibodies principally as described by Ganrot (1972). This method was used as a complement for the detection of contaminating antigens and for comparison between the different albumins as regards heterogeneity. Determination of the protein content was carried out according to Lowry eh al. (1951) with bovine serum albumin fraction V (Sigma) as standard. Gel-filtration In order to determine the mol. wts of the albumins, gel-filtration was performed at +4”C on a 2.5 x 80cm Sephadex G-200 column buffered with a 0.05 M Tri+HCl buffer and 0.15 M NaCl. nH 7.5. as described earlier (Carlsson ef ai, 1976). The me&od of Andrews (1965) was used. Soybean trypsin inhibitor (Sigma) mol. wt 21,500; ovalbumin (Grade-V, Sigma) mol. wt 45,000; bovine albumin (fraction V. Sigma) mol. wt 67,000 and 134,000 and anorfer;itin (Serva, Hiidelberg) mol. & 480,000 were used & reference proteins. One millilitre swine serum, 0.75 ml colostrum diluted with 2 ml elution buffer, 3 ml foetal serum and 30 ml urine ultra-coated to 3 ml were applied. The ~stribution of albumin was determined using ele~roimmunoassay and crossed immunoelectrophoresis. Hectrophoresis
in polyucrylamide
gels
Polyacrylamide gel-electrophoresis was performed on an Ortec annaratus in 0.065 M Tris-borate buffer., PH 9.0. __ using a gradiporegel 426% as described earlier (Car&son et al., 1976). L
Ultra-centrijkgation
The sedimentation coefficient was measured in a Spinco Model E Ultracentrifuge at 59,780 rev/min, 259,700 g as described earlier (Carlsson et al., 1976). The sedimentation coefficient s&,_ was calculated by extrapolating to zero protein concentration.
Amino acid composition
Amino acid analysis was performed with a Jeol-6AH automatic amino acid analyzer as described previously (Carlsson et al., 19’76). Isoelectric focusing Isoelectric focusing was carried out on a 110 ml LKB column for 90 hr with a voltage from 450-1000 V, at a pH range from 4 to 6 built up by Ampholine Carrier ampholytes (LKB). A sucrose density gradient was used as a stabilizing medium. The samples were collected in 2.5 ml fractions using an Ultrorac 7000 fraction collector. The pH was measured on a Bergman and Beving pH meter at the same temperature as that used during isoelectric focusing (+ 3°C). Eight milligrams of SS-albumin, FS-albumin. Col-albumin and neonatal serum albumin obtained after preparative electrophoresis and 8 mg urine albumin obtained from 30 ml neonatal urine concentrated by ultrafiltration to 2 ml were focused. Focusing was also performed on 8 mg SS-albumin and 8 mg Co&albumin pretreated with charcoal (Grave) at nH 3, &d at +4”C for 1 hr according to Chen (1967). The albumin distribution in the pH gradient was determined by means of electroimmunoassay. RESULTS adult urine
Isolation
of ul~u~~~ from
colostrurn
and neonatal
serum,
foetal
serum,
Despite, two electrophoretic runs on pevicone, the fractions of SS-containing albumin were contaminated with three other proteins. After a%inity chromatography on Con-A Sepharose which has the ability to bind g~ucoprote~s (page, 1973), two of these proteins were removed. By means of the ASS-immuno-
sorbent column, as described above, the last contaminating protein was removed and pure SS-albumin was obtained (Table 1 a-d and Fig. 1). When the purity of the albumin preparation was assayed by electro~unoassay, ~munoelectrophoresis and crossed immunoelectrophoresis with ASS as antiserum, only one precipitation arc could be seen (Fig. 1). In addition, electrophoresis in polyacrylamide gels showed one distinct band and immunization of rabbits with the isolated albumin resulted in a monospecific antiserum (Table le). Although the antiserum used for production of the ASSA-immunosorbent column was monospec~c when tested by immunoelectrophoresis it was not possible to obtain pure albumin after chromatography on this column. The preparations were found to be contaminated by minute amounts of one protein with the same electrophoretic mobility as that of albumin. This conta~nating protein was present in the prep~ations of albumin from SS, FS, colostrum and neonatal urine (Fig. l), However, it was possible to remove it by means of affinity chromatography on the ASS-immunosorbent column as described above. The albumins thus obtained were proved to be pure by the production of monospecific antibodies after immunization of rabbits. Moreover. in tests with electroi~~oassay, crossed immunoelectrophbresis and immunoelectrophoresis using antisera to adult serum, foetal serum and colostrum, SS-, FS- and CM-albumin gave one precipitation arc (Figs. 1 and 2). However, the albumin from urine showed a different pattern as was best seen by crossed immunoel~trophoresis where two peaks showing
ROLAND N.
288
K.
CARLSON, BENGT I. INGVARSON AND B~RJE W. KARLSON
Fig. 1. Elcctroimmunoassay showing the purification steps for adult swine strum albumin (A, B), foetal serum albumin (C), colostrum albumin (D) and urine albumin (E). Part 4 shows swine serum (a) and fractions obtained after preparative electrophoresis (b). Con-A affinity chromatography (c) and ASS immunosorbance (d). Parts BE show whole serum (c) and fractions obtained after ASSA immunosorhance (f) and fractions obtained after ASS immunosorbance (g). An antiserum to adult swine strum was used (0.03 ml/ml agarosc).
partial identity wcrc formed (Fig. 2D). These results were confirmed by clcctrophoresis in polyacrylamide gels. In addition to the one distinct band revealed by SS-, FS-, Col- and urine albumin the latter showed
IDF--lr----!
Fig. 2. Crossed immunoclcctrophorcsis on isolated albumins from adult swine serum (A), foetal serum (B), colostrum (C), neonatal urine (D) and neonatal urine (E) after addition of equal amounts of isolated albumin from swine serum, foctal strum or colostrum. In (F) the neonatal albumin protile of whole neonatal serum is shown. In gels A E the same picture was ohtaincd using antisera to adult swine strum. foctal serum, colostrum or adult serum alhumin. In gel F an antiserum to adult serum albumin was used. The dark spots in the lower left corner of the gels indicate sample application sites.
Table
2. Molecular
s&,,
Mol. wf und sedimentation corjficient The mol. wts and sedimentation coefficients (s&).~.) of the albumins arc listed in Table 2. From this table it can be seen that the differences in mol. wts found by gcl-filtration wcrc confirmed by the dctcrminations of the sedimentation coefficients. Urine albumin consists of two components with different mol. wts and sedimentation coellicients. When the fractions obtained after gel-filtration of the urine were tested by crossed immunoclcctrophorcsis it was seen that the larger
weight and sedimentation coefficient (s!, ,) of SS-albllmin albumin, Col-albumin and urine album&
SS-albumin Mol. wt
a second band (Fig. 3). This hctcrogcncity. further investigated by crossed immunoclcctrophorcsis. was found in both concentrated otherwise untreated urine and in the pure preparation of urine albumin. The proportion between the two types of albumin found varied between different samples. Addition of SS-, FSor Col-albumin to urine albumin resulted in enlargement of the slow moving albumin fraction while the fast moving fraction was unaffected (Fig. 2E). When serum from piglets aged 24-hr was tested no albumin heterogeneity could be observed (Fig. 2F). Addition of SS, I-S, Co1 or urine to the antisera against the various albumins yielded complete absorption as revealed by immunoclcctrophorcsis. In contrast to earlier findings (Carlsson er al., 1976) no Con-A reactivity of H-albumin could be seen.
FS-albumin
Col-albumin
urine albumin
67,500
66,500
65,000
4.58 + 0.05
4.55 * 0.08
4.52 + 0.04
a Due to small amounts
this value could
not be measured
65,500 44,500 4.46 & 0.19 3.1”
more exactly
W-
289
ide gradipors gel 4-26% run for 160 minutes. showing from Hi 1, . 1 . . . --_-..
291
Albumins from porcine serum, colostrum and urine
(b)
0 40
5.0
6.0
pH-values 4. Distribution of albumin after isoelectric focusing in the pa-range 4-6. Figure (a) demonstrates adult swine serum albumin (O-O), foetal serum albumin (A-A) and neonatal serum albumin (04). Figure (b) shows colostrum albumin (O---U) and neonatal urine albumin (A-A) while Fig. (c) shows charcoal-treated swine serum albumin (t-0) and charcoal-treated colostrum albumin (W---B). component (mol. wt 65,500) corresponded to the slow migrating fraction (first peak), while the smaller component (mol. wt 44,500) corresponded to the fast migrating fraction (second peak) (Fig. 2D).
Isoelectric focusing. The albumin distribution in a pH gradient from 4 to 6 after isoelectric focusing is shown in Fig. 4. SS-albumin and FS-albumin follow the same overall distribution pattern with major peak values around pH 5.5 (Fig. 4a), while colostrum and urine albumin have major peak values at pH 4.5 (Fig. 4b). The albumin from neonatal serum has two large peaks, one at pH 4.5 the other at pH 5.5 (Fig. 4a). It is notable that all the albumins except urine albumin have peak values around the same pH, e.g. 4.5, 4.9 and 5.5, but that the proportion between the peaks differs for the various albumins. Treatment of SSalbumin and Col-albumin with charcoal did not change the SS-albumin distribution significantly whereas the major peak of Col-albumin changed from pH 4.5 to 4.9 (Fig. 4~). Amino acid composition. The amino acid composition of the albumins is shown in Table 3. Almost identical values were found for Col-albumin and SSalbumin with minor variations in glutamine and histidine content. The amino acid composition of FSalbumin differs from that of SS-albumin and Colalbumin, major differences being seen in serine, proline, leucine and lysine content and minor differences in asparagine, valine, cysteine, tyrosine and phenylalanine content. The composition of urine albumin shows intermediate values between E&albumin and Col-albumin on the one hand and FS-albumin on the other for 9 amino acids, while 5 amino acids showed higher values and 3 lower values.
DISCUSSION The principle used for the isolation of swine serum albumin (SS-albumin) described in this paper involves the preparation of a crude fraction of albumin and subsequent chromatography on an immunosorbent column to which antibodies against swine serum (ASS) was coupled. Though tedious, this method can
Table 3. Amino acid composition of SS-albumin, Col-albumin, FS-albumin and urine albumin
Asparagine Tbreonine Serine Glutamine Proline Glycine Alanine l/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan
SS-albumin
Col-albumin
FS-albumin
urine albumin
84 43 38 169 55 29 83 58 59 8 34 93 33 50 100 20 45 not measured
84 44 39 159 55 29 82 61 62 6 36 93 31 49 95 30 43 not measured
75 39 50 170 90 35 16 50 51 5 31 79 42 58 85 24 39 not measured
94 41 41 163 63 44 78 54 58 10 34 84 29 46 86 31 30 not measured
The values are expressed as moles/1000 moles of amino acids.
292
ROLAND N. K. CARLSSON.BENGT I. INGVARSSONAND B~RJE W. KARLSSON
be valuable not only for the isolation of albumin but also other proteins provided that it is possible to obtain a crude preparation in which the protein in question is predominant. However, it is important to be aware of possible sources of error which may occur when the albumins from adult and foetal swine serum, colostrum and urine are isolated using antibodies against adult swine serum albumin in the ASSA-immunosorbent step. This method of preparation could cause a loss in specific albumin components as FS-, Col- and urine albumin may contain specific parts not common to the SS-albumin molecule. However, this risk must be considered to be merely theoretical as such a molecule must lack antigenic determinants present in the SS-albumin molecules. Moreover, the absorption studies, as revealed by immunoelectrophoresis, showed that the different isolated albumins were immunologically identical as the antibodies produced were common to the various albumins. The fact that chromatography on the ASSAimmunosorbent column did not render pure albumin seems remarkable as the antiserum (ASSA) used for preparation of the immunosorbent column proved to be monospecific by the immunological test methods used. It could depend on the presence of minute amounts of undetectable contaminating antibodies, or on binding of the contaminating protein to the fixed anti-albumin-antibodies or on unspecific binding of the contaminating protein to the Sepharose matrix. The results from the investigation of the physicochemical properties of the albumins indicate that differences do exist. These differences may be due to the environment in which albumins are found, or to the developmental stage of the animal or may reflect functional differences. The great similarities in amino acid composition between SS-albumin and Col-albumin indicate that Col-albumin is derived from the serum of the lactating sow. This is in accordance with earlier findings (Polis et al., 1950; Laurel1 & Morgan, 1965; Morgan, 1968; Jennes, 1974). An explanation of the differences noted between SS- and Col-albumin concerning mol. wt, sedimentation coefficient and isoelectric-focusing pattern must be sought in variations in conformation and bound substances such as fatty acids and bilirubin. The results from isoelectric focusing of charcoaltreated Col-albumin and SS-albumin support the assumption that there are differences in the fatty acids bound to Col-albumin and SS-albumin as the p1 pattern of charcoal-treated Col-albumin is strongly affected compared to that of native Col-albumin, whereas charcoal-treated SS-albumin is not significantly affected. The results on charcoal-treated Colalbumin are in agreement with those of Valmet (1969) and Rosseneu-Motreff et al. (1970) who found an increase in p1 after treatment of human serum albumin and bovine serum albumin with charcoal. However, Valmet states that fatty acid-albumin complexes are unstable below pH 5 and that the fatty acids are separated from the albumin molecule during the electrofocusing procedure. This could offer an explanation to the fact that charcoal-treatment of SSalbumin in our study did not affect the p1 as the focusing was run for 90 hr with a voltage of 1000 V during the last 60 hr, with a consequent separation
of the fatty acid-albumin complexes. As the same procedure was used for electro-focusing of Colalbumin we cannot conclude at the present stage that the p1 peak at 4.5 of Col-albumin is caused by the interaction of fatty acids, although treatment with charcoal affected the p1 in a way typical for defatted albumins. The amino acid composition of FS-albumin differs from that of SS-albumin and Col-albumin. This observation supports the findings of Miyoshi et al. (1966) who found a foetal type of human albumin (Alb F) different from that of the adult (Alb A). However, the differences in amino acid composition found in our study do not result in any changes in the p1 pattern of FS-albumin as compared to SS-albumin. This may depend on the fact that the major variations in amino acid composition occurred in non-polar and uncharged amino acids. Moreover it can be concluded that the variations in amino acid composition do not give rise to any immunogenic determinants not common to FS and SS as complete absorption was obtained both when anti-FS-albumin was absorbed with SS-albumin and vice versa. Bergelin and Karlsson (1975) stated that urine albumin from neonatal piglets has a mol. wt of 50,000. This value is based on tests with gel-filtration and subsequent electroimmunoassay. From the present work it is obvious that urine albumin is heterogeneous and consists of at least two components with mol. wts of 65,500 and 44,500. In order to visualize this heterogeneity, electroimmunoassay is an inappropriate test method and crossed immunoelectrophoresis must be used. It has been known for many years that intact proteins are absorbed via the gut of the neonatal pig (Nordbring & Olsson, 1957; Munn & Smith, 1974). Though Page (1969) assumed that the neonatal synthesis of albumin could be totally responsible for the observed abrupt rise in the concentration of albumin, Karlsson (1970) is of the opinion that absorption from colostrum is at least partly responsible for this elevation in concentration. The fact that it was possible to detect the major p1 peaks of both Col-albumin and FS-albumin in the p1 pattern of serum albumin from neonatal piglets aged 24 hr is interpreted to be due to the existence of albumin of both foetal and colostral origin in the neonatal piglet. Results from our investigation on neonatal urine albumin suggest that this albumin partly originates from colostrum. The p1 pattern is very similar to that of Col-albumin with one large peak at pH 4.5. Furthermore, both colostrum albumin and the large component of urine albumin have almost identical mol. wts (65,000 and 65,500) and the precipitates formed during crossed immunoelectrophoresis show a tailing towards the application very common for colostrum albumin, neonatal serum albumin and neonatal urine albumin. The larger component of neonatal urine albumin (mol. wt 65,500) is probably identical to that found in the serum of the neonatal piglet, since addition of SS-, FS- or Col-albumin to the samples or urine albumin as revealed by crossed immunoelectrophoresis increased the amount of the slow migrating fraction without the formation of new peaks. Moreover, the determination of mol. wt, sedimentation coeffi-
Albumins from porcine serum, colostrum and urine cient and the results from polyacrylamide gel-electrophoresis lend further support to this conclusion, The smaller component of neonatal urine albumin (mol. wt 44,500) cannot be detected in serum from piglets aged 24 hr. It seems likely that this component is a breakdown product of the larger one. Although our results indicate that the breakdown may occur in the kidney or in the urine, it cannot be excluded that the breakdown takes place in the intestine before or during the absorption of proteins. ~artinsson (1973) found that urine ~bu~n from neonatal piglets aged 12 hr consisted of 2 fragments and that addition of pure swine albumin revealed a new peak as seen in crossed immunoelectrophoresis. Our finding that intact albumin occurs in the urine can be explained by the fact that we used a pool of urine from piglets aged 10-24 hr. The results From ammo acid analysis on total urine albumin indicate that the albumins found in urine may originate from the foetus as well as from colostrum, as values intermediate to those of Col- and FSalbumin were found for 9 amino acids. For another 8 ammo acids values which were either higher or lower than the corresponding values for Col-albumin and FS-alb~in were found. A possible expl~ation for this deviation may be that the small mol. wt albumin component in neonatal urine is selected during the isolation procedure and has an amino acid composition different from that of the high mol. wt albumin component. The results from this investigation suggest that colostrum albumin originates from sow’s serum. After onset of colostrum ingestion this albumin is absorbed via the intestinal absorptive cells into the bloodstream of the newborn piglet. Therefore the albumin in the serum and urine of the newborn piglet after onset of colostrum ingestion is a mixture of albumin from colostrum and albumin synthesized by the piglet itself. We feel that research in the field on the various albumin types in the body fluids of the pig could give results which would be of use in the investigation of the function of, for example, the intestinal mucosa cells and the cells in the nephron in the kidney of the newborn tion.
piglet after the onset of colostrum
inges-
Acknowledgements-The skilful technical assistance of Mrs. Marie Adler-Maihofer and the secretarial help of Miss Marianne Andersson are gratefully acknowledged. We are also indebted to Dr. Ulla-Britt Hansson at the Biochemical Institute for performing the sedimentation analysis, and to Miss Ing-Britt Johansson at the University Hospital in Lund for performing the amino acid analysis. This investigation was supported by the Royal Physiographical Society, Lund. REFERENCES
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