9/6/2018 Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

Indian Journal of Biochemistry & Biophysics

Vol. 41, August 2004, pp. 148-153

Biochemical characterization of a protein of albumin multigene family from serum of

African catfish Clarias gariepinus Bloch
Absar-ul Hasnain1*, Riaz Ahmad1, Mumtaz Jabeen1 and M Mushahid Khan 2
1Biochemical
Genetics Lab., Department of Zoology and 2Interdisciplinary Biotechnology Unit,
Aligarh Muslim University, Aligarh 202 002, India
Received 17 September 2003; revised 27 June 2004

A monomorphic albumin-like protein (CfSA) has been purified to homogeneity from the serum of African air-breathing catfish
Clarias gariepinus Bloch. It has a molecular mass of @70 kD and shows a lesser electrophoretic mobility than human serum albumin
(HSA) in native gels. The protein exhibits cross-reactivity against rabbit anti-HSA serum and shows considerable similarity with HSA in
secondary structure, however, with some differences, as indicated by a slight shift in the peaks around 267 nm and 278 nm and the
absence of shoulders at 276 and 283. A certain degree of similarity also exists between their tertiary structures with respect to aromatic
asymmetric environment as indicated by far-UV CD spectra and the visible range CD spectra of bilirubin complexes. CfSA-bilirubin
complex is mainly characterized by bisignate CD Cotton effects (CDCEs), having minima and maxima wavelengths at 406 and 486 nm,
respectively and unlike HSA, it shows prominent additional maxima around 426 nm. Based on the number of sulfhydryls, CfSA is in the
rank of advanced teleosts. The occurrence of albumin in C. gariepinus in relation to the evolutionary dichotomy of albumin and other
members of its multigene family in class Pisces has been discussed.

Keywords: Biochemical characterization, ligand transport, catfish serum albumin (CfSA), human serum albumin (HSA), lipoprotein,
α-foetoprotein, sulfhydryls, circular dichroism, bilirubin binding, helix topography, Clarias gariepinus.

Serum albumin has apparently evolved from an ancestral protein which diverged into two other lineages, one leading to
evolution of α-foetoprotein (AFP) and the another, to vitamin D-binding protein (VDBP)1,2. As a protein that transports
wide range of physiological ligands, albumin is ubiquitous in higher chordates3,4. However, in fishes, occurrence of
albumin appears to be selective and in some cases, it is replaced by lipoprotein, from the multigene family. More than one
type, albumins, albumin-like proteins or lipoproteins might exist together in some cases, which appear in accordance with
the ontogenetic and developmental schedules. In class Pisces, albumin has phylogenetic significance. For instance,
Petromyzon marinus, one of the most primitive living chordates possesses an oversized serum albumin (175 kD) that
possibly diverged from an ancestral form, prior to the gene duplication that gave rise to AFP and VDBP5. Two albumin-
like proteins AS and SDS-1 have also been purified from P. marinus6. On the basis of sequence homology with
mammalian albumins, AS is considered an early version of AFP7. Moreover, during the life cycle of parasitic8 and a non-
parasitic lamprey (Lampetra appendix)9, relative amount of AS has shown variation with the developmental stages. It is
reported to be completely absent in the adult L. appendix9.
It is also intriguing that despite a general trend of increase in the amount of albumin with the evolutionary advancement, it
is altogether absent in elasmobranches10,11, which are more advanced than cyclostomes. Among bony fishes, several species
lack albumin. The polyploid salmonids contain albumin12 or related protein13,14, whereas in the Antarctic toothfish
(Dissostichus mawsoni)15, eels (Anguilla dieffenbachii and Anguilla australis schmidtii)16 and the mirror carp (Cyprinus
carpio)17, the ligand transport is taken over by a high density lipoprotein having binding properties similar to albumin. Also,
two 70 kD albumin-like proteins which cross-react with anti-HSA (human serum albumin) antibodies and a co-existing
lipoprotein of 130 kD have been reported from the rainbow trout Salmo gairdnerii14,18.
Taking into account the restricted occurrence of albumin or another member of multigene family, information on
teleosts as the group of highest evolutionary diversity among vertebrates, is far from adequate. Earlier, in an air-breathing
fish Channa gachua, we demonstrated the presence of an albumin-like protein, which exhibits polymorphism controlled by
two co-dominant alleles, cross-reacts with anti-HSA antibodies and binds with Cibachrome blue dextran19. In the present
study, we attempted to purify an albumin-like protein, referred to as catfish serum albumin (CfSA) hereafter, from the
serum of the evolutionary distinct African walking catfish Clarias gariepinus, which diverged from the main lineage of the
teleosts as a specialized group of obligate air-breathers. The protein was characterized by (i) determining molecular mass
by exclusion chromatography on Seralose-6B and SDS-PAGE; (ii) immuno-blotting; (iii) comparison of sulfhydryls; and
(iv) studying the CD-spectral properties to elucidate secondary and tertiary structures of free and bilirubin-protein
complexes.
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9/6/2018 Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

Materials and Methods

Human serum albumin (HSA), bovine serum albumin (BSA), ribonuclease, myoglobin, ovalbumin, catalase, γ-globulin,
DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), sodium dodecyl sulphate (SDS), Coomassie brilliant blue R-250 (CBB),
ammonium persulphate, bromophenol blue (BPB) and ammonium sulphate were obtained from Sigma-Aldrich Chemical
Co., USA. Seralose-6B was from LKB-Pharmacia, Sweden and bilirubin was obtained from Sisco Research Laboratories,
India. Buffer salts used in the experiments were of analytical grade.
Collection of fish samples and isolation of serum
Live C. gariepinus in the size range of 25-30 cm during July-August were obtained from local fish market and
acclimatized for 2 days in laboratory aquaria. Out of 50 specimens, 40 were females.
Fishes were anaesthetized by triacane-methosulfate and the blood from heart was collected using sterilized syringe and
transferred to sterilized glass test tubes. It was allowed to clot at room temperature and stored at 15°C. After 4 hr, serum
was collected with the help of microsyringe and centrifuged at 1500 ´ g to remove suspended blood cells.
Electrophoresis
Individual sera samples were screened by native PAGE, carried out in the system of Laemmli20 with the modification
that all gels contained 10% glycerol but no SDS, to exclude probable polymorphism. However, for monitoring purity,
unmodified protocol of SDS-PAGE was employed. Bands were developed by staining with Coomassie brilliant blue.

Purification and general biochemical methods

Albumin-like protein (CfSA) was isolated from the pooled serum by (NH4)2SO4 fractionation. Serum was saturated
with 2.26 M (NH4)2SO4, pH 7.0 (adjusted with NH4OH) and kept for 12 hr at room temperature. Following centrifugation
at 10,000 ´ g, the precipitate was discarded and pH of clear supernatant adjusted to 4.2 with 0.5 N H2SO4. The CfSA was
precipitated by bringing down molarity of (NH4)2SO4 to 1.9 M with careful addition of distilled water. Again, the
precipitation was allowed to proceed for 12 hr, centrifuged as above and the precipitated pellet was washed with 2.2 M
(NH4)2SO4 solution, pH 4.2 and dissolved in a small volume of 0.06 M phosphate buffer, followed by exhaustive dialysis
against 0.06 M phosphate buffer (pH 7.0). Final purification to homogeneity was carried out by Seralose-6B gel
chromatography21.
Protein concentration was determined according to Lowry et al.22, using BSA as the standard. The Stoke’s radius of the
isolated protein was determined by analytical gel filtration as described earlier21 and treated according to the straight line
equation23: Stoke’s radius (nm) = 9.74, erfc Kd +0.78.

Circular dichroism spectroscopy

CD spectra measurements were made on a Jasco spectropolarimeter, model J-720 equipped with a microcomputer. The
instrument was calibrated with d-10 camphorsulphonic acid. All the measurements were carried out at 25°C with the help of a
thermostatically-controlled cell holder attached to a Neslab’s RTE-110 circulating water bath with an accuracy of ±0.1°C. The
far-UV CD spectra were taken at a protein concentration of 1 mM (for fish serum protein) and 2 mM (for HSA) with a 10 mm
path length cell. Spectra were recorded at a scan speed of 20 nm/min and with a response time of 1 sec. Each spectrum is the
average of 4 scans. In the near-UV region, CD spectra were measured at a protein concentration of 5 mM with a 10 mm path
cell. Visible range CD spectra were measured at 350-550 nm at 25±0.1°C using a cell holder of 10 mm path length. The
spectra were collected at a scan speed of 500 nm/min with a response time of 1 sec. Each spectrum is the average of 3 scans.

Estimation of thiols
Thiols were estimated spectrophotometrically at 412 nm by incubating with DTNB according to Ellman24. Incubation
was carried out in 8.0 M urea, a concentration where HSA is fully unfolded25. The values represent average of duplicate
determination of two different preparations. The molar values calculated according to Ellman were converted into moles
SH/mole albumin taking Mr of both albumins as 70 kD.

Binding of bilirubin to albumin

Bilirubin binding experiments were performed in 0.06 M sodium phosphate buffer, pH 8.0 ionic strength 0.15. Bilirubin
solution was prepared by dissolving 5 mg solid bilirubin crystals in 1 ml of 0.5 M NaOH solution containing 1 mM EDTA
and was diluted immediately with the above buffer. The concentration of bilirubin was determined spectrophotometrically,
using a molar absorption coefficient of 47,500 M-1 cm-1 at 440 nm26. CD spectral measurements of bilirubin-albumin

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9/6/2018 Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

complex were made after incubating bilirubin-albumin solution for 30-40 min at 25°C. The spectra were recorded in dim
yellow light to prevent undesired photo-degradation of bilirubin.
Results
An albumin-like protein, referred to as catfish serum albumin (CfSA) with a molecular mass of @ 70 kD has been
purified from the serum of the African catfish C. gariepinus and studied for its biochemical and immunological properties.

Electrophoresis of serum albumin

Typical patterns of serum sample alongwith the purified protein in SDS-PAGE are shown in Fig. 1a which demonstrates
the degree of purity and similarities in the molecular mass of CfSA and HSA. Immuno cross-reactivity of the purified
preparation against rabbit anti-HSA serum is demonstrated by Western blots of SDS-PAGE patterns (Fig. 1b). Weak
signals by the patterns of whole serum (Fig. 1b, lane 1) indicate quite a low concentration of the protein. The molecular
mass of @ 70 kD, as determined by SDS-PAGE is further supported by gel filtration data along with various marker
proteins on Seralose-6B, that gives a value of Stoke’s radius of this protein as 3.34 (Fig. 2).

Fig. 1—(a): SDS-PAGE patterns of whole serum of Clarias gariepinus (lane 1), partially purified CfSA (lane 2), pure HSA, (lane 3), highly purified
CfSA (lane 4), and molecular weight markers (lane M); (b): Scan of western blot of SDS-PAGE patterns developed by LumiGlo fluorescence
(Loading sequence of the lanes 1-4 the same as in 1a). Gels (10%) were made in Tris-HCl and run in Tris-glycine essentially as described by
Laemmli’s (1970). For Western blots, SDS-PAGE patterns were transferred onto the PVDF membranes (Millipore, USA). Immuno-cross-reactivity
was determined using rabbit anti-HSA serum as primary antiserum. Goat anti-IgG coupled with HRP (Horse Radish Peroxide) was used as secondary
antiserum and detecting the cross-reactivity in Western blots with LumiGLO chemiluminiscent kit (KPA, USA).

Fig. 2—Gel filtration plot showing Stoke’s radius of catfish serum albumin-like protein (CfSA) [erfc-1 is the inverse error function complement of
Kd. Determinations were made on Seralose-6B column (80 ´ 1.15 cm) using the following markers proteins: 1, ribonuclease; 2, myoglobin; 3,
ovalbumin; 4, bovine serum albumin; 5, catalase; and 6, γ-globulin. The position of CfSA is indicated by arrow]

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9/6/2018 Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch
CD measurements
The secondary structure of CfSA was studied using CD measurements made at pH 7.4 (Fig. 3A). The far-UV CD
spectra of CfSA are characterized by two minima at 212 nm and 222 nm, comparable with the minima of HSA at 208 nm
and 222 nm, characteristic of an a-helix structure. The near-UV CD spectra of HSA and CfSA (Fig. 3B) revealed tertiary
structure at pH 7.4. In CfSA, a significant deviation in the positions of minima was observed towards higher wavelength
side which are positioned around 267 nm and 278 nm, respectively (Fig. 3B). It is interesting to note that shoulders at 276
nm and 283 nm are absent in CfSA. The main contribution from tryptophanyl residues to the observed ellipticity in the
near-UV CD region was obvious at 251, 268 and 284 nm, whereas that from tyrosyl at 277 nm and from phenylalanine at
251, 261 and 268 nm. It thus appears that all the three aromatic chromophores contribute significantly to the observed
ellipticity in the near-UV region of CfSA. The near-UV CD spectra of HSA is recognized by the presence of two minima
at 262 nm and 268 nm and two shoulders at 276 nm and 283 nm, characteristics of aromatic amino acids asymmetric
environment.

Fig. 3—Far (A) and near UV-CD spectra (B) of purified human serum albumin (HSA, thin line) and albumin-like protein of C. gariepinus (CfSA,
thick line). [The concentration for far UV-CD was 2 mM and 1 mM, respectively. For recording near UV-CD spectra, the protein concentration of both
albumins was 5 mM. Measurements in 10 mm path-cell were made in thermostatically controlled cell holders at 25°C. Each spectrum is the average of
4 scans].

Sulfhydryl contents
Following incubation in 8.0 M urea (final concentration), titrable thiols were calculated to be ~25 and ~33 SH
mole/mole of CfSA and HSA, respectively.
Bilirubin binding to CfSA and HSA
Visible region CD spectra of bilirubin complex with CfSA and HSA measured in 0.06 M sodium phosphate buffer, pH
8.0 are shown in Fig. 4. The spectra of CfSA-bilirubin complex (Fig. 4A) resembled bilirubin-HSA complex. In either
case, CD spectra are characterized by bisignate CD cotton effects (CDCEs), having minima at shorter wavelengths and
maxima at higher wavelengths (Fig. 4B). However, the bilirubin-HSA complex has shown an additional prominent CDCE
around 426 nm.

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9/6/2018 Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

Fig. 4—A comparison of visible range CD spectra of bilirubin-protein complexes (A): of CfSA; and (B): HSA [The concentrations of CfSA and HSA
were 1.5 mM and 10 mM, respectively. 5 mg solid bilirubin crystals were dissolved in 1 ml of 0.5 M NaOH solution containing 1 mM EDTA and
immediately diluted with 0.06 M sodium phosphate buffer, pH 8.0 and ionic strength 0.15. Other details are given under ‘Materials and Methods’]

Discussion
Albumin, following its divergence from the ancestral gene, has an evolutionary history of 400 million years11 and share
several characteristics with other proteins of common origin and evolution. Due to functional overlaps with sister proteins
of the multigene family, albumin cannot be unambiguously identified by any single criterion. As the above results
demonstrate, a number of criteria have to be applied to elucidate structural and functional properties of CfSA.
The similarity in size and shape of CfSA and HSA is evident from the value of Stoke’s radius (3.34 for CfSA), which is
in good agreement with the reported value of 3.56 for BSA and HSA21,27. Further, the Mr value of @70 kD obtained by
SDS-PAGE (Fig. 1A), is similar to that obtained for HSA and also for salmonid serum albumins12-14.
As judged by relative intensity of fluorescence signals in Western blots, CfSA shows considerable immunological
similarity with HSA (Fig. 1B). However, as compared to mammalian sera, where albumin is one of the major constituents,
a striking feature of CfSA is its extremely low amount (<0.5 %) in the serum, which is even lower than that reported in
rainbow trout14 and the Atlantic salmon (Salmo salar)28. In Western blots, CfSA gave strong signals only up on
employing highly sensitive fluorescence LumiGlo detection, while cross-reactivity of salmonid albumin was stainable with
4-chloro-1-naphthol14. However, unlike albumin-like proteins of salmonids14, 28 and C. gachua19, CfSA is neither
dimorphic nor polymorphic. Further, despite belonging to an evolutionarily distinct group of obligatory air-breathers,
biochemical profile of CfSA is substantially different from that of the cyclostomes6-9.
CD spectra report on the secondary structure of albumin-like protein or its internal environment in fish is hardly
available. In the present study, CD spectra results on CfSA, however, showed a general similarity between the molecular
architecture of CfSA and HSA, however, with minor differences in their secondary and tertiary structures (Fig. 3). As
compared to 208 nm of HSA, a shift in far-UV CD spectra to 212 nm is characteristic of CfSA, indicating more conserved
helix topography of two serum albumins29. The occurrence of two deep minima, at 267 and 278 nm in CfSA and also in
HSA at 262 and 268 nm with two shoulders at 276 and 283 nm are the characteristics of aromatic amino acid asymmetric
environment. While the above features indicate similarity, differences in tertiary structure of CfSA were evident from a
deviation in position of minima towards higher wavelengths of 267 and 278 nm and lack of shoulders at 276 and 283 nm.
Notwithstanding, a very long intervening evolutionary history between teleosts and mammals, the helical topography of
CfSA and HSA appears to be considerably conserved and hence, wide differences may not exist at the level of tertiary
folding and aromatic asymmetric environment.
CD spectra of bilirubin-CfSA complex showed similarity as well as differences with those of bilirubin-HSA complex.
Though albumins from both the sources bound bilirubin in a folded conformation with positive chirality, characterized by
bisignate CD cotton effects (CDCEs), an additional positive CDCE around 426 nm for bilirubin-CfSA complex suggests
conformational differences between its tertiary structure and HSA. Interspecies variations between CD values of bilirubin-
albumin complexes are attributed to differences at their binding sites30. Analysis of amino acid composition showed that
methionine and cysteine are the main residues which discriminate mammalian from fish albumins12,14. The occurrence of
lesser SH groups (~25 moles) in CfSA, as compared to HSA (~33 moles) is consistent with the trend in evolutionary
advancement of vertebrate sera albumins predictable on the basis of cysteine residues14.
Serum albumin-like proteins from the rainbow trout and the Atlantic salmon are reported to bind with palmitate and
bromocresol green (typical ligands of albumin), but not with Cibachrome Blue F3GA or 8-anilino-1-naphthalene
(ANS)14,28, but the co-existing lipoprotein of rainbow trout binds with the ANS18. However, the CfSA differs from these
serum albumin-like proteins and lipoprotein in binding with Cibachrome blue F3GA column and elutes out with gradient,
resembling a similar protein isolated from C. gachua19.
The findings suggest that the African walking catfish C. gariepinus may be categorized with the few known teleosts,
where the evolutionary divergence and selection has favoured retention of serum albumin as the transport protein of
circulatory system.

Acknowledgement
Facilities provided by the Aligarh Muslim University, Aligarh are gratefully acknowledged. Thanks are due to Dr Saad
Tayyab of Interdisciplinary Biotechnology Unit for his help and critical review of the manuscript and to Dr Rizwan Husain
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Khan for additional cooperation.

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________________
*Corresponding author
E-mail: zyt04ah@amu.ac.in
Abbreviations: CD, circular dichroism; CDCE, circular dichroism cotton effects; AS protein, ammoecoete serum protein; SDS-1 protein, sodium
dodecyl sulphate-1 protein; AFP, a-foetoprotein; HSA, human serum albumin; CfSA, catfish serum albumin.

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