STUDIES ON THE HAEMOGLOBIN OF ISOPARORCHIS HYPSELOBAGRI
DISSIRTAnON SUPIHTTBP IN PARTIAL FULFILMENT FOR THE PEOREE OF
MASTER OF PHILOSOPHY
BY
KHWAJA AFTAB RASHID
SBCnON OF ?AR^S\TOLOOY DEPARTMENT OF ZOOLOGY
ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)
4 I ,S 173^ . f,
'SUA J i .J -
DS1734
A l l ^ a p H M u s l i m XJzi i vex*B i t y
Ather H. Siddiqi Ph.D. (Alig.), Ph.D. (Purdue) Professor of Parasitology
CERTIFICATE
SECTION OF PARASITOLOGY
DEPARTMENT OF ZOOLOGY ALIGARH. U. P. 202 001
I certify that the work presented in this
dissertation has been carried out by Mr Khwaja
Aftab Rashid and that it is suitable for the
award of the M.Phil., degree in Zoology of
the Aligarh Muslim University, Aligarh.
Ather H. Siddiqi Supervi sor
DEDICATED TO MY PARENTS
CONTENTS
PAGE NO.
ACKNOWLEDGEMENTS (i)
INTRODUCTION AND HISTORICAL REVIEW 1
OBJECT OF THE STUDY 18
MATERIALS AND METHODS 21
RESULTS 38
DISCUSSION 45
REFERENCES 53
- 1 -
AOCNOW LEDGEMENTS
I wish to put on record my most sincere thanks and immense
gratitude to Prof. Ather H. Slddlql , my supervisor for helping me to
conceive this problem and extending all-round help in the execution of
this work. His cr i t ical insight, helpful suggestions and the efforts to
inculcate a scientific temper in me is gratefully acknowledged,
I am especially grateful to Dr. Masoodul Haqiie and Dr. Jawed
Siddiqui for their manifold help and encouragement during the course of
this study.
A special thank is also due to Dr. A.M. Kidwai, Industrial
Toxicologica] R e s e a r c h C e n t r e , Lucknow for a l l o w i n g me in h i s
l a b o r a t o r y to p u r s u e some of t he e x p e r i m e n t s .
Financial assistance in the form of a Junior Research Fellowship
from the University Grants Commission under Departmental Research
Support Programme [Project No. F-ll-18/87(SR-Ij ] is gratefully
acknowledged.
(KHWAJA AFTAB RASHID)
INTRODUCTION AND HISTORICAL REVIEW
The term haemoglobin was assigned by Hoppe-Seyler
(1864) more than one hundred years ago in relation to the
pigments of the blood. Lankester (1868) suggested the word
"erythrocruorin" (red pigment) as a general term for
protohaem oxygen binding respiratory pigments without
accounting for the structure or function of the pigment.
Analogous pigments of certain marine worms were termed
"ch lorocruorins". Smith (1961) named erythrocurorin all
those known or presumed protohaem respiratory pigments,
which are capable of undergoing reversible oxygenation,
while those erythrocurorins known or presumed to function
in oxygen storage, were classified as myoglobins.
Myoglobin is a relatively small, oxygen binding protein
(16,700 MW) found in muscle cells. Wayman (1966)
suggested that the role of myoglobin within cells" is
the counterpart of haemoglobin at the macroscopic level
of transport in the whole organism, the only difference
being that in one case the driving mechanism is a pump (the
heart), in the other a molecular process (translationa 1
diffusion)". All myoglobins and alpha and beta chains of
all modern haemoglobins share six invariant residues and
have many other positions in which closely related amino
acids are found. X-ray diffraction and chemical analyses
of haemoglobin and myoglobin structures have revealed that
these two proteins have similarity in tertiary structure.
-2-
this is probably also true for ery throcurorins
(myoglobins). These facts are suggestive of the common
evolution of myoglobin and haemoglobins from an ancestral
oxygen binding haem protein. Korbar (1866) reported
differences in the patterns of denaturation of haemoglobins
of various species by strong acids or alkalis. This
species specificity has been confirmed by modern
physicochemica 1 and immunological, techniques.
Investigations on the porphyrins by Kuster (1912),
(Wi 1 Is tS Iter et al'«'1913) and by various workers, were
particularly important in complete synthesis of protohaems
(Fisher and Zeile, 1929). It has been demonstrated that
iron atoms in both reduced haemoglobin and oxyhaemog lobin
are in the same electronic condition, the divalent or
ferrous state. They become oxidized to the trivalent, or
ferric state if haemoglobin is treated with a ferricyanide
or removed from red blood cells (RBCs) and exposed to air
for a considerable time. The studies on the chemical
derivatives of haem (Haurowitz, 1928) strongly suggested
that the prosthetic groups of all haemoglobins are
identical. Polderman (1932) arrived at similar results
on the basis of spectroscopic studies. Shortly thereafter
Roche and co-workers (1934) provided conclusive proof for
this assumption by demonstrating the differences in the
amino acid content of various animal haemoglobins. The
first end group analysis of haemoglobin was performed by
-3-
Porter and Sanger (1948). They showed that in human and
horse haemoglobins the terminal amino group was contributed
by valine. In beef, sheep and goat haemoglobins, the
terminal amino groups were valine and methionine. These
qualitative results have been confirmed by other workers
also. In 1938, a Cambridge group of crysta1lographers
began their work on haemoglobin using X-rays to provide
information about the external and internal structure of
the protein molecule. After many setbacks, a fundamental
breakthrough was achieved in 1954, which led towards an
understanding of the protein structure (Green et a 1.. 1954).
Four years later, a moelcular model of myoglobin had been
derived (Kendrew et al., 1958) and two years after that,
a similar model was available for horse haemoglobin. Thus
the X-ray data provided a picture of the structure of
haemoglobin which was entirely consistent with the
information obtained by chemical and physical techniques.
-4-
Structure : Wherever there Is need for oxygen in the living
organisms, it is transported by a conjugated oligomeric
chromopro_t.giJL, the haemoglobin. Haemoglobin consists of
a large protein molecule, the globin, a histone consisting
of four polypeptide chains (two alpha and two beta), to
each of which is attached a prosthetic haem group. Haem
is based on a structure known as a porphyrin ring which
includes four pyrrol groups around a central ferrous iron
(Fe * ) . The iron is joined by four of its coordination
bonds to nitrogen atoms of the porphyrin and by two bonds
to imlda/.o 1 nitrogen atoms contained in histidine residues,
within the protein globin.
The detailed three dimensional structure of
haemoglobin was revealed by X-ray analysis by Perutz
(1978). The haemoglobin is roughly spherical molecule with
a diameter of about 5,5 nm. Each of the four chains has
a characteristic tertiary structure, in which the chain
is folded. The alpha and beta chains of haemoglobin
contain several segments of alpha helix separated by bends.
The four polypeptide chains fit together in an
approximately tetrahedral arrangement, to constitute the
characteristic quaternary structure of haemoglobin. There
is one haem group bound to each chain. The haems are
rather far apart, about 2.5 nm from each other, and tilted
at different angles. Each haem is partially hurried in
a pocket lined with hydrophobic 'R' groups. It is bound
-5-
to its polypeptide chain through a coordination bond of the iron atom
to the 'R' group of a histidine residue, the sixth coordination
bond of the iron atom of each haem is available to bind
a molecule of oxygen. There are many contact points
between the alpha and beta chains of the dissimilar chain
pairs oc e and 00262 ^^d little direct contact between
the two alpha chains or between two beta chains. Theoc rj
and 0C262 pairs are made up of irregularly shaped
polypeptide chains, these do not fit with each other
precisely. There is a central open channel or cavity
running right through the haemoglobin molecule, which can
be seen in the top view of the molecule.
Both X-ray diffraction and chemical analysis of
haemoglobin structure, have revealed an important set of
relationships. First, it has been found that the alpha
and beta chains of haemoglobin have nearly identical
tertiary structures. Both have well over seventy percent
alpha helical character, similar lengths of alpha helical
segments, and the bends or turns have about the same
angles. Second, the hemoglobins of many different
vertebrate species have approximately the same tertiary
structure of their polypeptide chains. Moreover, the
quaternary structures of different haemoglobins closely
resemble each other. The third important point is that
the tertiary structures of the alpha and beta chains of
-6-
haemogloblns are very similar to the tertiary structures
of myoglobins. Thus the similar tertiary structures of
myoglobin and alpha and beta chains of haemoglobin can be
related to the capacity of both proteins to bind oxygen
as their biological function.
The family of relationships between myoglobin and
haemoglobin chains is further shown by a comparison of the
amino acid sequences. Sperm whale myoglobin and alpha and
beta chains of horse haemoglobin show twenty seven
identical residues in cpmparable positions and have very
closely related res'fdues in fonty aCher pdsitLons.. Thus
amino acid sp<(aences of homologous proteins share a number
of coTf^ponding invariant residues and that homologous
proteins tend to have similar three dimensional structures.
Occurrence and distribution of haemoglobin The pigment
haemoglobin is very common and wide spread in animal
kingdom. Spectroscopica 1 ly haemoglobins have been detected
in microorganisms such as ascomycetes fungi, Neurosgora
crasga and Peni ci 2_ll_um noj^a^um (KeiUn and Tissieres, 1953 );
among protozoans P3Ii[Dece|um cauda^um and Jslrahi^mena
pyrl formls (Keilin and Ryley , 1953) possess haemoglobin
like pigments.
An oxygen binding pigment, identical with the human
haemoglobin has also been found to occur, in root nodules
-7-
of leguminous plants and referred to as leghaemog lobin
(Sternberg and Virtaney,1952). These help in oxygen supply
for the bacteria symbiotic in the root nodules for nitrogen
fixation, therefore, leghaemog lobins are helpful in
symbiotic relationships, though exceptions do exist.
The leghaemog lobins are resolved
chroma tographically into two major and two minor components
of molecular weight 17,500 and 19,500 respectively.
Distribution of respiratory pigments was reviewed
by Fox and Vevers (1960) in invertebrate animals. Amongst
the members of the P latyhe Iminths , i t has been found in the
representatives of the classes Turbellaria, Trematoda,
and Monogenea but is definitely absent in class Cestoda.
In phylum Nematoda, different haemoglobins occur in
perienteric fluid and bodywall.
In annelids the physiological and chemical
properties of haemoglobins have been studied in quite
detaiL These show striking differences in structure and
oxygen equilibrium properties (Manwell, 1960; 1963; 1964*,
Jones 1963; Florkin, 1969) they range in size from high
molecular weight, extracellular haemoglobins
(erythrocruorins) to low molecular weight, intracellular
haemoglobins and myoglobins (Svedberg, 1933; Svedberg and
Eriksson-Quense 1, 1934; Hoffman and Mangum, 1970; Mangum,
1970; Weber,1972; Terwilliger and Koppenhoffer, 1973).
-8-
Dissolved haemoglobin is found in the blood of some
members of the entromostrace Crustacea (Fox, 1957). Among
the insects, haemoglobin is somewhat rare in occurrence,
being found only in fevv Diptera and Hemiptera.
Haemoglobins are also found in blood of some gastropod
molluscs and some echinoderms and limited studies have been
made of their properties e.g., by Yagi et a 1., (1955) on
the molecular weight, absorption spectra and amino acid
composition of Andra ln _la_ta
Most invertebrate haemoglobins are extracellular
and possess comparatively high molecular weights with lower
isoelectric points compared with the intracellular
haemoglobins of vertebrates. However, haemoglobins of
relatively low molecular weights are found in Chi_ronomus,
some annelid species, some nemerteans and in the
lame 1libranch mollusc A£ca. In both, Area and the
polychaete N2l2!Di£lH£« ^^^ haemoglobin with a molecular
weight of about 30,000 is contained within the corpuscles.
These low molecular weight haemoglobins can be contrasted
with the haemoglobins of invertebrates such as Daghni a
(360,000 MW) and Pianorbj.s carneres (300,000 MW) ( VVyman,
1948). The random distribution and wide diversity in the
nature of invertebrate haemoglobins implies an independent
line of evolution of this protein.
-9-
In vertebrates the haemoglobin is the only respiratory
pigment in the blood. In most vertebrates the haemoglobin
is tetrameric, each molecule consisting of four globin
chains with each chain associated with a haem group.
(Brannitzer, 1958; MliUer, 1961 a,b). The mass of
vertebrate haemoglobin ranges from 61,000 to 72,000 but,
despite considerable difference in the primary structures
of their globin chains in higher vertebrates, the
isoelectric pH is restricted to a range of 6.9 - 7.3 and
in lower vertebrates to the approximate pH range of 6.0
- 8.0 (Gratzer and Allison, 1960).
Myoglobin, another oxygen binding protein,
occurring in the muscles of vertebrates, is a monomeric
form. It is quite probable that myoglobin, a single
polypeptide chain protein, and haemoglobin, a tetrameric
protein, could have evolved from a common ancestral oxygen
binding haem protein, which may have had but a single
polypeptide chain. At some point in further evolution of
species the gene coding for the ancestral oxygen binding
protein may have become duplicated. These two gene copies
then underwent mutations independently, so that one of them
gradually coded for the myoglobin type of protein, adapted
to storage of oxygen in cells, and the other gene underwent
a different pathway of mutation to code ultimately for the
alpha and beta chains of haemoglobin, adapted to
transportation of oxygen in the red blood cells.
-10-
The primitive haemoglobins still consist of only
a single peptide chain. The haemoglobin of Lamp^era has
a molecular weight of 17,000 and possesses only one haem
group, thus implying it to be monomeric. Svedberg (1933)
found that haemoglobins of molecular weights of 17,000 and
34,000 were found in l Z lHi SiHlill°§§. suggesting the
presence of both monomers and dimers.
Haemoglobin in helminths : Apart from haemoglobin, no
ott»er respiratory pigments (such as haemocyanin,
haemerythrin or ch lorocruorin) have been detected in
parasitic helminths. Wherever examined in detail, the
parasite haemoglobins are always distinct from those of
their hosts (Lutz and Siddiqi, 1967; Haider and Siddiqi,
1977 and Fusco, 1978). The haemoglobin in parasitic
helminths occurs in the tissue, but in nematodes it is also
found in solution in the perienteric fluid. Occasionally,
haemoglobin is located preferentially in certain regions
of the parasite e.g., in Fasc2.oJ.a hega_ti ca, it is found
primarily around the vitellaria and uterine coils, and in
the nematode, ^erm_is subni.g^rescens, it is the pigment in
the anterior chromotrope (light sensitive organ). The
association of haemoglobin with the vitellaria in F.
l25E£li£5 "' y be related to the oxygen requirement of egg
tanning. Stephenson (1947) found abundant haemoglobin near
-li
the viteUaria and distal uterine coils in F. he£at^i.ca,
whereas Lutz and Siddiqi (1967) observed concentrations
around both suckers of Fasci oJ a £is.§![lii£i • Cain (1969a)
located haemoglobin in P J.2og_thaJ.arnus !5§Saili£H§ ^"^
Ei5£i£i°Esis buski_ throughout the parenchyma with noticeably
greater intensity near the proximal uterine loops and
bordering the excretory spaces, less colour was evident
in the suckers and parenchyma adjacent to the vitellaria
and jCQca , Caln/also detected host haemoglobin in the gut
of P. [negajurus, but not in F. buskj., although virtually
absent from the reproductive organs, a positive benzidine
reaction was observed in developing eggs of P. !5ega_lurus.
Parasites frequently contain multiple haemoglobins.
In the^/Trematoda, the haemoglobins of F. aiaaQllHa-
2i£I2£2§iiH!2 ^£!l^£i.li£M!2 • £• !D§fiiiH£H£ ^^^ §£!}i£££i£!I]2
££^£iHiHn! ^^^ b^ separated into two components, but F.
buski^ has only one haemoglobin. The perienteric fluid and
body wall haemoglobin of Ascari^s iH[Dbri_coJ^des may be
further separable into two components. The gapeworm,
§y£Si!Bli£ !£§£!}§£• ^^s only one haemoglobin (Rose, and
Kaplan, 1972) but there are three haemoglobins in
l£l£5!D££££ £2!ll££§> five in Qs tert agi a sp., and six in
5^£iis£oides £££i££ii.- The body wall of 0. £££i££J:i
contains three haemoglobins, the perienteric fluid two and
there is one in the gut. Developing fourth and fifth-stage
•12-
larvae of 0. cuni_cuj.i_ have been reported to contain yet
anottier different haemoglobin. In T. conl^sa, only the
females have haemoglobin and in Sgi_rocart|a_12anus cri_cot^us,
the haemoglobins from males and females have different
isoelctric points (Fusco, 1978).
The free living stages of parasitic nematodes,
however, do not appear to contain haemoglobin and in the
development of 0. £yili£uj.i^, haemoglobin first becomes
detectable in the parasitic fourth stage larva.
Haemoglobin is found in larval Tri^chi^neJ^^i §.Ei£§.ii§. ^^^
larval Eust^rong^yji^des iS.not^us but both of these larvae are
tissue parasites. Vandergon et al., (1988) detected
intracellular haemoglobin in gymnophallid metacercariae
parasitic in the metanephridia 1 sacs of the common marine
worm AmEhil£ile orna^a a polychaete, the haemoglobin of
these metacercariae show characteristic absorption spectra
for oxygenated, deoxygenated and carbon monoxide
derivatives and had an oxygen half saturation (P^^) value b U
= 1.1. The pigments also showed cooperative oxygen binding
with a Hill coefficient of 2.2 and exhibited a significant
Bohr effect between pH 6.8 and 7.4
With the exception of A. iumbri.coi_des the physical
properties of parasite haemoglobin have not been
extensively studied. The only values so far available for
the molecular weight of helminth haemoglobins are: F.
fl££ali£§ 17,500; F. buski 15,000-, D. dendr|_t i.cum 15,500-,
-13-
S. llichea 38,400 and A. iHS^£i££i55£• perienteric fluid
haemoglobin 328,000^ body wall haemoglobin 40,600.
Generally, in invertebrates, intracellular haemoglobins
have a relatively low molecular weight, whereas
extracellular haemoglobins have a high molecular weight.
Oxygen affinity of parasite haemoglobins : An important
parameter of haemoglobins is the oxygen affinity. The
oxygen tension at half saturation, the ?_„ is generally
used as an index of oxygen affinity.
All of the parasite haemoglobins show a high oxygen
affinity and there may be an inverse relationship between
P-^ and environmental oxygen tension, the mammalian gut
parasites all having a low P-n. whilst S. trachea (from
the trachea of birds) and Cama^l^anu^ llls2i!22sus (from
reptiles) have higher P__, values. In A. lumbricoides the
perienteric fluid haemoglobin has a higher oxygen affinity
than the body wall haemoglobin. Usually, where two
haemoglobins are present in an animal, the reverse
situation exists, with the tissue haemoglobin having the
highest affinity, thus enabling oxygen to be transferred
from the circulating fluid to the tissues. The P_„ = 0.01 5 0
mmHg at 37°C of A. iH[I!^£i££i^5s perienteric fluid
haemoglobin for oxygen is the highest reported for any
haemoglobin and is a reflection of the extremely small
dissociation constant .Tuchschmid et al., 1978 and recently
-14-
Smi t et al., (1986) observed a high oxygen affinity in case
Shape of the oxygen dissociation curve : In mono haem
pigments, such as tiyo g lobin, the oxygen dissociation curve
is hyperbolic. In multihaem pigments, the haem groups can
interact with one another and the oxygen dissociation curve
becomes sigmoidal. All of the parasites haemog lobins so far
studied have not shown haem-haera interaction. Some
parasite haemoglobins, such as the one from A. llJi2bri_coi des
body wall, are monohaem pigments, but others, like
perienteric fluid haemoglobin, are multi-haem pigments.
Bohr effect : In many invertebrate haemoglobins there is
little or no Bohr effect and in some cases a reverse Bohr
effect has been observed, i.e., acidification increases
oxygen affinity. In parasitic helminths, the haemoglobin
°^ §• i£§£!}£^ shows a small positive Bohr effect, whilst
the body wall and perienteric fluid haemoglobins of A.
iy!D^£i£2i^25. show a small reverse Bohr effect. A reverse
Bohr effect may be of adaptive value for an animal living
in an environment low in oxygen and high in carbon-dioxide.
TuchsdTiad et al., 1978; Smi t et al., 1986; have observed
a marked acid Bohr effect in the liver fluke Di,crocoe2i.um
dendri t icum.
-15-
Physiological role of haemoglobin in helminths : Oxygen
binding pigments, such as haemoglobins, have two distinct
functions. They either help to maintain a continuous supply
of oxygen to the tissues or they act as an oxygen reserve.
In addition. Intracellular haemoglobins can facilitate the
passage of oxygen through tissues at low oxygen tensions.
The haemog lobin- modulated facilitated diffusion of oxygen
through a static solution is called the Scholander effect
(Scholander , 1960). It may be an important function of
invertebrate tissue haemoglobins.
All of the helminth haemoglobins that have been
studied in detail have high oxygen affinities and probably
remain fully oxygenated In vivo. These haemoglobins also
become deoxygenated under anaerobic conditions, except the
perienteric fluid haemoglobin of A. l!J[2brj coi_des which can
be deoxygenated only by chemical means (dithionite
treatment).
Physicochemical properties of trematode haemoglobins :
Spectral Properties : The spectral study of haemoglobin
in trematodes has been made by many workers such as
Wharton (1939, 1941): van Grembergen (1949); Goi1 (1959,
1961); Freeman (1963); Todd and Ross (1966); Lutz and
Siddiqi (1967); Cain (1969b)and Haider and Siddiqi (1976).
The absorption spectra of the porphyrins are so
characteristic that in many cases they can be used for
-16-
identifying and differentiating their kinds. This is
especially true for haemoglobin and its derivatives which
can be classified as ferrous ionic or ferric covalent
compounds, according to the nature of their absorption
spectra, Haider and Siddiqi (1976) studied the spectral
properties of several derivatives of haemoglobins of six
different trematodes from three different hosts and
concluded that porphyrin IX is the common prosthetic group,
and oxyhaemog lobin, carbon monoxy haemoglobin and reduced
haemoglobin gave absorption maxima similar to those of the
equivalent haemoglobins of their hosts. Pronounced
differences were observed, however, in the spectral
behaviour of cyanmethaemog lobin and pyridine derivatives
when compared to similar derivatives of their respective
host haemoglobins.
Alkali denaturatlon : Exhaustive studies were performed
by Haider and Siddiqi (1977) on the susceptibility to
alkali denaturatlon of the haemoglobin in digenetic
trematodes, which showed that trematode haemoglobin is more
resistant to alkali denaturatlon than their corresponding
host haemoglobins, however, the pattern of denaturatlon
varies among the trematodes. This was probably due to
variations in the amino acid sequences of a particular haem
protein. A Ithough de tai led information on the amino acid
-17-
sequence and crystal structure of haemugloDln of D.
®Il £ili£H[II ^s available but not published (Smit, 1983).
Nuclear magnetic resonance (NMR) studies by Lecorate et al.,
(1987, 1989) suggest the distal residue to be a tyrosine.
Blectrophoretlc properties : Lutz and Siddiql (1967)
first of all demonstrated the difference in the
e lectrophoret ic pattern of F. fiifi.ailli25 ^^^ ^^^ host
haemoglobin using paper electrophoresis. Cain (1969b)
determined the molecular mass of F. buski_ haemoglobin by
sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) system, Cain also studied the e leetrophoretic
behaviour of F. buski_ haemoglobin in aery lamide gels with
and without urea and proposed that F. buski_ haemoglobin
is a monomeric protein which is consistent with its
molecular weight and single haem content.
OBJECT OF THE STUDY
The mysterious presence of the respiratory pigment and
its intriguing function in trematodes, which are mostly metabolically
anaerobic, has perplexed scientists for long.
The vertebrate haemoglobins and myoglobins have been
comparatively well studied. While most of the studies on trematode
haemoglobins have been limited to the identification of the pigment,
estimation of the amount present and i ts distribution among
trematodes. The precise physicochemical properties and
physiological role of haemoglobin in trematodes h&ve been far less
thoroughly investigated.
The trematode haemoglobin has always been suspected of
its endogenous origin and having a function useful for the trematode.
This is because the parasite obtains i ts nutrition from the host
tissues, part of which consists of host blood. The parasite thus
probably acquires haemoglobin from the host either in part or as
a whole molecule. Moreover, the trematode, being an anaerobe,
does not require oxygen for respiratory metabolism (oxidative
metabolism).
However, the spectrophotometric, electrophoretic and
oxygen binding properties of trematode haemoglobins have
demonstrated that they are different from their respective host
haemoglobins. This suggests that parasites may be synthesizing
these oxygen binding coloured proteins and utilizing thorn in their
physiology.
-19-
It is imperative to know the physicochemical properties
before studying the functional aspects of these unique oxygen
binding proteins.
The haemoglobin molecules present one of the most
rewarding instances of studying biological phenomenon, such as
the evidences on the phylogenetic relationships of the trematodes.
Trematodes and chordates have been evolving along independent
Unas probably since the Cambrian times. Differences between
helminth and vertebrate haemoglobins might .therefore, be expected
to be considerably larger than vertebrate haemoglobins and
myoglobins.
Study of widely different type? of haemoglobins is
desirable if the relation between structure and function of
haemoglobin molecules is to be understood, such a study will allow
coprelation of structural features possessed by different
haemoglobins with properties peculiar to each.
The detailed study of haemoglobin molecules may help
us in understanding the Influence of niche segregation and niche
adaptation at molecular level, as there is no other molecule more
versatile than haemoglobin which responds to the changes according
to the physiological needs of the organism in which it is found.
The biological significance of these pigments in anaerobic
trematodes would also help in the better understanding of the
intrinsic physiological relationship between host and parasite,
•20-
and their comparative physiology and biochemistry. Such studies
would also be helpful to trematode taxonomists in providing a
more reliable basis for characterizing the trematodes at species
level.
MATERIALS AND METHODS
The trematode isogarorchis l}ZE£®l25iS£i ''^
collected from the swim bladder of the catfish, VVaj.j.ago
a _tu, obtained from the local fish market. In the
laboratory the fish trematodes were washed several times
with fish normal saline and incubated in the same saline
with 8 mM glucose for one hour at 30 ± 2°C, so as to
shed their eggs and eliminate hematin from their ceca.
The parasites were then damp dried and stored in a deep
freezer at -20°C until sufficient material was
CO 1 lee ted.
The host haemoglobin was obtained from the blood
collected in 1:1000 heparin (used as an anticoagulant)
by bleeding live fish through a cut at tail end. The
heparinized blood was centrifuged at 2000xg for 10 min
at 4°C to separate plasma. The RBCs so settled were
washed three times with isotonic saline (0-9% NaCl, 0.15
M) and then lysed in an equal volume of double distilled
water. After haemolysis, cell stroma was separated from
the solution by centrifuging the haemolysate at 10,000xo
for ten minutes. The pure haemoglobin solution was then
dialyzed and concentrated against polyet.hy lene glycol
(20,000 MW), lyophilized and stored for future use.
The trematode haemoglobin v/as extracted by
homogenizing the parasites and precipitating the
-22-
proteins by ammonium sulfate, in the following manner : The
parasites were homogenized in an ice cold mortar with a pestle
in throe volumes of 0.06M phosphate buffer pH 7.5, to
which 5 mM of ethylenediamine tetraacotic acid(EDTA),
ImM of phenyImethyIsulfony 1 fluoride (PMSF) were added
as proteolytic enzyme inhibitors. The tissue debris
was removed by centrifuging the homogenate at 16,000xg
for 15 min in a ref erigera ted centrifuge, the pellet
so formed vvas discarded and the supernatant was
saturatad to 40% of ammonium sulfate concentration.
After one hour the precipitated proteins were
^ontrifuged at 15,000xg for 15 min and the ammonium
sulfate concentration of supernatant was increased to
70%. The precipitated proteins were again separated
by centrifuging at 15,000xg for 15 min at 4° C and a
final concentration of 95% ammonium sulfate was
achieved at which the haemoglobin was completely
precipitated. The same was left overnight at 4°C . The
precipitated haemoglobin was dlalysed in 2-3 changes
of 2000 volumes of 0.06M phosphate buffer, pH 7.4 for
12 hours to remove ammonium sulfate. Dialysis was
carried out in a dialysing bag No. D-9527, Sigma
Chemical Co. Further purification and molecular mass
was determined by gel filtration chromatography.
-23-
Gel filtration chromatography : This technique was
employed to further purify the partially purified
trematode haemoglobin and estimate its molecular weight.
Gel filtration is advantageous in that it does not
require highly purified protein for the determination of
molecular weight.
Packing of the column : A slurry of Sephadex G-75 was
made and suspended in enough 0.06 M phosphate buffer pH
7.4, It was then allowed to swell overnight at room
temperature. A glass column (2.5 x 100 cm) (Pharmacia
CIO) was used for gel filtration. Before pouring the gel
into the column it was deaerated under vaccum to remove
trapped air bubbles. A gel reservoir was fixed on the
top of the column so as to achieve a 95 cm of gel bed
height. The gel was allowed to settle under gravity for
twelve hours and a flow rate of 15ml/hr was gradually
achieved using a peristaltic pump (Pharmacia P-1). The
even packing and void volume of the column were checked
by watching the passage of blue dextran through it.
Column calibration : The Sephadex G-75 (2.5x95 cm)
column was calibrated by determining the partition
coefficient (Kav) for proteins of known molecular
weights (Schachraan, 1963) : Bovine serum albumin
-24-
(67,000 MW); ovalbumin (43,000 MW) and myoglobin (17,000
MVV) , and calculated from the formula.
e - 0 Kav = -Y~Z~y~~
t o
where V is the elution volume, V the void volume e 0
(elution volume of blue dextran ) and V. the total
volume of the gel bed (Andrews, 1964).
The calibration curve for the column shows a
linear relationship between Kav values and molecular
weight standards (Fig.l ).
Application of the Sample : The haemoglobin sample
before being loaded onto the column was equilibrated
with the elution buffer by dialyzing it for 12 hrs
against several changes of the buffer. The sample was
applied by adapter AC 10 supplied by Pharmacia with its
C 10 column. The adapter allows the even application
of the sample in a fast and reproducible manner without
causing disturbance to the bed surface.
Fractions of 5 ml each were collected by an
automatic fraction collector (Pharmacia Frac 100) at a
flow rate of 15 ml/h. The absorbance of each fraction
was measured at 280 nm and 412 nm on a (Spectronic 21)
spec tropho tometer.
Figure 1 : Calibration curve for the estimation of molecular weight of proteins by Sephadex G-75 gel filtration chromatography.
IS
- 2 5 -
o
^ i
Ae>
-26-
The elution volumes of partially purified
haemoglobins of Iso^arorchis hj^gse^oba^ri^ and its host
\ iiiiS.° iiiH were then measured and the molecular
masses were estimated by extrapolating the Kav values
calculated by the formula as proposed by Andrews (1964).
The two fractions obtained (Fig. 2 ) in the
case of ifogarorchi^s h^2§£i25i&£i partially purified
haemoglobin were pooled separately, dialyzed to remove
salts, concentrated against polyethylene glycol (which
due to its high molecular weight does not enter Into
the dialyzing bag) and lyophilizod.
Absorption maxima : The absorption maxima (Fig. 3 )
of the haemoglobin fractions obtained by gel filtration
chromatography was obtained by scanning the pooled
samples from 400 nm to 600 nm v/ave length at the
intervals of 10 nm wavelengths.
Protein 9stimatlon : Protein estimation was carried
out by the method of Bradford (1976) and Spector (1978)
using 'Spectronic 21' spectrophotometer. The protein
concentration was read on a previously established
standard curve, prepared by using bovine serum albumin (Fig. 4).
Figure 2 : Sephadex G-75 gel filtration of proteins precipitated from homogenate of Isoparorchis hypselobagri by 70-95% ammonium sulfate saturation.
- 2 7 -
7.
in d
AllSNad IVJU.Kl
Figure 3 : Absorption maxima of Fraction-I and Fraction-II obtained by Sephadex G-75 gel filtration, of proteins from homogenate of Isoparorchis hypselobagri precipitated by 70-95% ammonium sulfate saturation.
i
o
AiiSNao nvDUdO
Figure 4 : Calibraticn curve for the estimation of proteins by the method of Bradford (1976) and Spector (1978).
-29-
m en
o
LT)
o CN
c
As
_ O
ID
' 1 00
o
1 r o
1 vO
O
1 m o
1 >3-
O
1 - CO
o
1 CN
O
mu s6g XV AXisNaa ivoixdo
-30-
Principle : Coomassie blue dissolved in acid at a pH
below 1 turns brown red, but when it binds to a protein
the blue colour is restored due to a shift in the pka
of the bound Coomassie blue, and according to the
protein concentration.
Procedure : The Bradford reagent was prepared as
follows. 100 mg Coomassie brilliant blue G-250 was
dissolved with vigorous agitation in 50 ml 95% ethanol,
then mixed with 100 ml 85% phosphoric acid. The mixture
was diluted to 1 litre with double distilled water and
filtered to remove undissolved dye. The solution was
kept for 1-2 weeks in cold.
To 0.1 ml of sample containing up to 50 ^g
protein, 2.5 ml of Coomassie blue reagent was added and
absorbance read at 595 nm after 2-30 minutes.
Simple electrophoresis : Simple po lyacrylamide gel
electrophoresis was performed to assess the homogeneity
and the differences in the banding patterns of the host
and parasite haemoglobins. Simple eleetrophoresis does
not interfere with the three dimensional structures of
proteins, i.e. does not denature proteins. For
electrophoresis, purified and lyophilized haemoglobins
-31-
from fish and its trematode ( !_. hZE^e_lobagri^) were
dissolved in sample buffer at a cone, of 2 mg/ml.
The electrophoresis apparatus v/as obtained from
Atto Corporation Japan. The Pharmacia (ECPS 3000/150)
A.C. mains power pack was used fqr constant current
power supply.
Prior to electrophoresis, the glass plates were
washed with a detergent rinsed with double distilled
water, dried and wiped clean with absolute alcohol.
Electrophoresis was performed in 15%
po lyacry lamide slab gel of (130x138x1mm) dimension
consisting of two sections (i) large pore spacer gel
in which proteins were stacked (ii) a small pore gel
in which e leetrophoretic separation was accomplished.
The discontinuous system, introduced by Laemmli (1970)
for disc gel electrophoresis, which was later adopted
to slab gels by Studier (1973), was employed.
This system is characterized by a discontinuity
in the buffer pH and in the po lyacry lamide pore size.
Electrophoresis was carried out at a constant current
of 20 mA for 3-4 hours, till the bromopheno1 blue used
as a tracking dye reached within 2-3 cm from the bottom
of the gel. The slab gel containing two sets of
haemoglobin samples was cut into two halves. One half
-32-
was stained overnight for protein with Coomassie
brilliant blue, destained by immersing in destaining
solution for 1 hour and subsequently washed in 10%
acetic acid. The other half was stained for haemoglobin
with benzidine reagent v/hich was prepared as follows.
A solution of 16 g of sodium acetate in 100 ml
of 7% acetic acid was saturated with ethylenediamine
tetraacetic acid (EDTA). After filtration, this
solution was saturated with benzidine compound and
refiltered. Immediately before use 0.1-0.2 ml of 3%
hydrogen peroxide was added to 10 ml ' of benzidine
reagent (Ornstein, in communication to Canal Industrial
Corporation). The haemoglobin bands appeared green or
greenish blue which turn brown in about twenty four
hours.
Sodium dodecyl sulphate polyacrylamide gel
electropharesis( SDS-PAGE) : It was employed to study the
molecular weight, subunit structure and purity of the
haemoglobin samples.
It has been empirically observed that the
electrophoretic mobility of the protein in a
polyacrylamide gel is inversely proportional to the
logarithm of its molecular weight. Thus a reasonably
accurate value for the molecular weight of a protein
can be obtained by comparison of their e lectrophoretic
mobility with known standards.
-33-
The proteins, when heated in the presence of
an anionic detergent - sodium dodecy I sulphate and a
reducing agent, are denatured and broken into the
subunits which are separated according to the
e leetrophoretic mobility.
If the protein is pure, only one band appears
in SDS-PAGE, otherwise more than one band ps indicative
of impurities. However, proteins having more than one
subunit type may resolve into multiple bands which are
not necessarily an indication of the presence of
impurities, if this is suspected then there should be
a rational relationship between the intensities of the
multiple components.
SDS-PAGE was carried out in (130x138x1 mm) slab
gels of 15% acrylamide and 0.1% SDS, for 3-4 hours at
20 mA per slab gel. The position of the tracking dye
bromopheno1 blue was marked by inserting a black nylon
bristle at the buffer front, SDS slab gel was immersed
in fixative solution for at least 10 hours, and several
changes of fixative were made to remove SDS. Overnight
staining was carried out in 0.25% Coomassie blue in
fixative solution. Gel was destained by soaking in
fixative solution for one hour and washing subsequently
by several changes of 10% acetic acid.
-34-
Preparatlon of the sample : The lyophilized samples
of parasite and fish haemoglobins were dissolved in 2x
sample buffer diluted 1:1 with distilled water to give
1 mg/ml protein concentration. These were heated in
boiling water bath for two minutes (Sigma Technical
Bulletin No. MWS-877L).
Preparation of SDS aolecular weight markers: SDS
molecular weight markers MW-SDS-70L Kit (Dalton Mark
VII-L) was obtained from Sigma Chemical Company.
Containing mixture of the following seven proteins, used
to plot calibration curve.
Proteins Approx. MW
i) Albumin bovine 66,000
ii) Albumin ^gg (Ovalbumin) 45,000
iii )G lycera Idehyde^-S-phospha te 36, 000
dehydrogenase. Rabbit muscle
iv) Carbonic anhydrase, 29,000
Bovine erythrocytes
V) Trypsinogen, Bovine pancreas 24,000
(PMSF Treated)
vi) Trypsin inhibitor. Soyabean 20,100
vii ) a-Lacta Ibumin, Bovine Milk 14.200
-35-
The contents of the vial of SDS molecular
weight markers were reconstituted in 1.5 ml of Ix sample
buffer and incubated at 37°C for two hours prior to
electrophoresis. Aliquots were frozen at -20°C for
future use. Ten micro litre of sample was used.
Calibration curve for estimation of molecular weights
by SOS-PAGE : The relative mobility (R.) of a protein
was determined by dividing the migration distance from
the top of the separating gel to the centre of the
protein band by the migration distance of the
broraophenol blue tracking dye from the top of the
separating gel.
f ' distance of tracking dye migration
The R. values (abscissa) are plotted against the
known molecular weights (ordinate) on semi-logarithmic
paper (Fig. 5 ) The molecular weights of unknown
proteins were estimated from the calibration curve.
Figure 5 : Calibration curve using Sigma SDS tnolecuJar weight markers (Dalton Mark VII-L), for the estimation of molecular weight by SDS-PAGE.
- 3 6 -
y -
- >
W 6oT
-37-
Purification of Isoparorchls hypselobagri haemoglobin.
Table - 1
Fraction Volume A412/A280 Fold Purification
Homogenate
40% Ammonium sulfate saturation
70% Ammonium sulfate saturation
95% Ammonium sulfate saturation
500ml 0.677
500ml 0.810
500ml 0.950
25ml 1.683
1.19
1.40
2.48
Fraction I obtained by Sephadex G-75 gel filtration
Fraction II obtained by Sephadex G-75 gel filtration
30ml 0.29
60ml 3.57
0.42
5.27
The starting material was lOOg of fresh frozen paras i tes .
Protein impurity not related to haemoglobin•
-38-RESULTS
Purification of haemoglobin : The results of purification
of haemoglobin are summarized in Table-1.
Most of the haemoglobin present in the homogenate
was recovered in the supernate. Ammonium sulfate
fractionation at 40% saturation yielded a 1.19 fold
purification, at 70% ammonium sulfate saturation 1.40, and
2.48 fold increase in purification was obtained at 95%
ammonium sulfate saturation. The haemoglobin obtained at
95% ammonium sulfate saturation was further purified by
Sephadex G-75 gel filtration chromatography, which resolved
the partially purified haemoglobin into two fractions.
Fraction I with a low ratio (0.42) of optical density at
412 nm and 280 nm was considered as a protein impurity,
while the ratio of the second fraction was close to that
of typical haemoglobin. There was a 5.27 fold increase
in the purification of parasite haemoglobin after gel
filtration chromatography. The molecular mass of the two
fractions separated on Sephadex G-75 column was estimated
to be 66 kDa and 17 kDa for fraction I and II,
respectively, while the molecular mass of the host, catfish
(Wallago attu) was 32 kDa. (Fig. 6 ).
Absorption maxima : The pooled haemoglobin fractions of
!• hypse lobagri showed characteristic absorption peaks of
soret, 6 and oc bands at 412, 540 and 560 nm wavelengths.
Figure 6 : Sephadex G-75 gel filtration of haemoglobin of Wallagu attu (catf ish).
.39-
o o
AJ.ISNSO nVDlXdO
•40-
Slmple Electrophoresis : Five microgram of host (Wa llago
attu) haemoglobin resolved into three sharp closely
migrating bands nearer to the cathodal end which was stained
with benzidine reagent and Coomassie blue. Five microgram
of fraction I of parasite haemoglobin after simple
electrophoresis appeared as a single sharp band towards the
cathodal end, when stained with Coomassie blue, however,
staining with benzidine required the application of 10 ug
of protein. Fifteen microgram of fraction II of partially
purified haemoglobin resolved into four major bands closer
to anoda1 end, which stain with both Coomassie blue and
benzidine reagent. Benzidine reagent also revealed three
minor additional bands. The first major band was heavily
stained by Coomassie blue than with benzidine reagent
(Figure 7a, b).
SDS-PAGE ; The results of SDS-PAGE of two fractions of
parasite haemoglobin and host haemoglobin are shown in
Fig. 8. The lane 'a' is the molecular mass standard.
Lane 'b' is fish haemoglobin where only one band
of ca.l4 kDa was observed. The second fraction of
Isoparorchis hypselobagri haemoglobin in lane 'c' provided
a single band of molecular mass ca. 15 kDa while the
fraction 1 in lane 'd' revealed five bands of molecular mass
ca. 12,000; 15,000; 35,500; 44,000 and 66,000 kDa,
respectively. The first and second bands were
diffused. Together with the fact that A412/A280
Figure 7a: Simple PAGE pattern in 15% acrylamide gel stained with benzidine reagent.
a) Wallago attu (catfish) haemoglobin, amount applied 5 pg.
b) Isoparorchis hypselobagri, Fraction I, amount applied 10 ^ g .
c) Isoparorchis hypselobagri haaicg Idbin, Rnaction II, amount applied 15 /ig.
- 4 1 -
a b c
ii
Figure 7b: Simple PAGE pa t te rn in 15% ac ry l amide ge l s ta ined with Coomassie blue reagen t .
a) I s o p a r o r c h i s hypse lobag r i Fraction I I , amount app l ied 15 ^ g .
b) I s o p a r o r c h i s h y p s e l o b a g r i , Fract ion I , amount appl ied 5 pg.
c) Wallago at tu (ca t f i sh) haemoglobin, amount app l ied 5 >Jg.
- 4 2 -
^M-
'-St.
Figure 8 : SDS-PAGE pa t te rn in 15% acry lamide ge l :
a) Sigma Dalton Mark VII-L, 14,000 to 66,000 MW; amount appl ied 10 u l .
b) Wallago at tu (ca t f i sh) haemoglobin, amount app l ied 10 yig.
c) I s o p a r o r c h i s h y p s e l o b a g r i , Fraction II; amount app l ied 10 j jg.
d) I sopa ro rch i s h y p s e l o b a g r i , Fract ion I , amount app lied 25 pg .
- 4 3 -
a b
5
4
-44-
ratio for fraction 1 was 0.42, it was safe to assume that
this fraction does not represent a haemoglobin.
Often difficulties were experienced in obtaining
clear electrophoretic pattern in SDS-PAGE system, of
haemoglobin which was extracted without the addition of
proteolytic enzyme inhibitors. Reasonably improved results
were obtained when 1 mM phenyImethyIsuIfony 1 flouride
(PMSF) and 5 mM Ethylenediamine tetraacetic acid (EDTA)
were added before homogenizing the parasites.
The proteinases responsible for the obscurity of
SDS-PAGE patterns of Isoparorchis hypselobagri haemoglobin
were assumed to be either serine esterases, thiol proteases
or some carboxy peptidases because these are inhibited
by PMSF or meta1 loproteinases which are inhibited by EDTA.
DISCUSSION
Purification of haemoglobin : Partial purification of Ascaris
perienteric fluid haemoglobin was first reported by Devenport
(1949). Attempts at further purification have been made
by Haraada et al., (1963) and Smith et al., (1963), but full
purity of the protein was not attained in either of these
studies. Wittenberg et al., (1965) purified Ascari.s
perienteric fluid haemoglobin by ammonium sulfate
fractionation up to 73% saturation and employing ion
exchange chromatogrpahy they obtained a 94% pure pigment.
No further improvement in final purification was obtained
by additional procedures tried, including gel filtration
on Sephadex G-lOO and G-200 or Amberlite IRC-50 column
chromatography. Cain (1969b) obtained a five fold
purification of haemoglobin from F. buski_ after partial
purification through ammonium sulfate fractionation (which
yielded a 3,8 fold purification) combined with preparative
disc electrohoresls which yielded samples over 95% pure,
as determined by densitometry of analytical gels. Cain
(1969 b) also reported that rechroma tography of the
fractions obtained by gel filtration chromatography over
Sephadex G-lOO did not give any improvement, nor did ion
exchange chromatography on DEAE Sephadex A-50. Instead,
the later procedure reduced eleetrophoretic mobility of
haemoglobin in acrylamide gels, presumably by altering
the size and/or charge of the pigment and at least three
-46-
other proteins. Recently Vandergon et al., (1988) purified
a haemoglobin from a gymnophalid metacercaria by employing
high performance liquid chromatography (HPLC) gel
filtration and ion exchange chromatography. They obtained
two fractions of haemoglobin referred to as A and B in
whole animal lysate. While ion exchange chromatography
resolved whole animal lysate into four components, the
ion exchange chromatography of the two fractions A and B,
obtained by HPLC gel filtration chromatography, resulted
in incomplete resolution of two components in each of the
above fractions which probably correspond to the component
I and II in fraction A and component III and IV in fraction
B.
The author purified the haemoglobin from
I§2E§£°££l}i§. l}ZES§i°^§S£l ^" which the pigment is present
in the body tissues, by precipitating the proteins from
whole animal homogenate by ammonium sulfate saturation up
to 95% in three broad cuts. The partially purified
haemoglobin (2.48 fold purification) was further purified
by gel filtration chromatography over Sephadex G-75 which
resolved the partially purified haemoglobin into two
fractions at 67,000 daltons and 17,000 daltons.
The degree of purity at each step was determined
by ratio of optical densities at 412 nm and 280 nm. The
above ratio of optical densities being very low for
-47-
fraction one, suggests that it is a protein irapyrity not related to
parasite haemoglobin. The second fraction possessed the A412/A280
ratio close to that of a typical haemoglobin.
Proteolytic enzymes have been reported from some
trematodes (Rupova and Xeilova , 1979* Simpkin et al., 1980,
Hamajuma and Yamagami, 1981: Chapman and Mitchell, 1982)
(Howard et al.i 1980) suspected that thermostable
proteolytic enzymes might degrade surface components of
F. hgBalica. In the present Investigation as well the
addition of proteolytic enzymes inhibitors in homogenate
have shown markedly improved results indicating the presence
of certain proteolytic enzymes in I. hi[gse2obagri_.
Absorption maxima : The absorption peaks of the parasite
pigment at characteristic wavelengths of haemoglobin,
quite firmly identifies the parasite pigment as
haemoglobin.
Simple electrophoresis : A protein molecule in solution
at any pH other than its isoelectric point has a net
negative charge. This causes it to move in an applied
electric field. The force is given by E, the electric
field (Vm ) times z, the net number of charges on the
molecule. This force is oppos forces in the
-48-
tnedium, proportional to the viscosity T^ particle radius
r (Stokes radius), and the velocity v; in a steady state:
Ez = 6 ir^ rv
The specific mobility u = v/E is given by
u = BTTt r
The difference in the eleetrophoretic pattern of
the host and parasite haemoglobin was first demonstrated
by Lutz and Siddiqi (1967) using paper electrophoresis.
As described in the results the eleetrophoretic pattern
of (2- llZE§.5l2 §.8.£i haemoglobin also differs from that
of the host (Wa^Jago a_t u) haemoglobin. The two fractions
obtained after the purification of parasite haemoglobin
through gel filtration chromatogrpahy were studied
separately for their e lectrophoretic mobility and banding
pattern. The first fraction appears as a single sharp band
while the second fraction resolves into at least four
major bands visible as distinct red bands in unstained gel
and by Coomassie blue staining. Benzidine treatment
revealed three additional faint bands near the major ones,
however, band 4 was deeply stained by Coomassie blue than
by benzidine reagent.
-49-
The only band of fraction I of X- hy EseJ-Oba ri.
haemoglobin stains deeply by Coomassie blue than by
benzidine reagent, probably because of higher content of
protein other than haemoglobin. By comparing the gels,
stained for total protein by Coomassie brilliant blue and
specifically for haemoglobin by benzidine, the homogeneity
of the parasite and host haemoglobin could be assessed,
as there were similar patterns of bands resolved by using
both types of staining.
Otherr workers like Lutz and Siddiqi {4r9B7) and Cain
(1969a) have also reported higher mebility in the parasite
haemoglobin than the host h rtnog lobin. However, F. busk^
haemoglobin and pig m^a^obin are almost identical in this
respect (Cain 195!
These results indicate that the trematode
haemoglobin may closely resemble vertebrate myoglobin and
suggested a study of molecular weight and subunit structure
of the chromoprotein purified.
Sodium dodecyl sulphate polyacrylamlde gel electrophoresis:
When proteins are heated with sodium dodecyl sulphate,
(SDS), (an anionic detergent,) and reducing agents, they
unfold, break into subunits and are almost totally
denatured. Moreover, the relationship between
electrophoretic mobilities and molecular weights of various
-50-
marker proteins of known molecular weights can be used to
approximate the molecular weight of an unknown protein.
The molecular weight is one of the fundamental
property of any protein molecule. Unfortunately the
molecular weights of haemoglobin of trematodes have been
determined only in few species and our knowledge in this
field of study is still fragmentary. The only values so
far available for the molecular weights of helminth
haemoglobins are 13,000 for F. buski. determined by Cain
(1969b). Tuchschmid et al., (1978), using gel filtration
in 6 M guanidine and SDS gel electrophoresis, reported the
molecular weight of D. dendri _t i cum haemoglobin as 15,500.
Barrett (1981) reported the molecular weight of F. hega_ti ca
haemoglobin to be 17,800. Among nematodes the molecular
masses of perienteric fluid haemoglobin from S. i£ichea
and A. iurahri coi des were reported as 38,400 and 328,000
respectively by Rose and Kaplan (1972). Vandergon et al.-,
(1988) found a molecular mass of 16,000 daltons in a
gymnophallid metacercaria while its annelid host Amghi__tri _te
orna_ta possessed a molecular mass of 3x10 and 16,000
daltons in vascular haemoglobin and coelomic cell
haemoglobin, respectively.
Haque et al., (in press) estimated the molecular
mass of purified haemoglobin fractions from Gas ro_t h^ lax
crumeni_£er and Pi£a!3Ehi_s_t omum egi.c2ilum by gel filtration
-51-
and SDS-PAGE. In SDS-PAGE system the two haemoglobin
fractions from G. crumeni f er were of 15 kDa, while a single
haemoglobin fraction from P. e£l£iiium was of 16 kDa.
However, the masses estimated by gel filtration were about
30 and 18 kDa in G. crumeni.£er and 16 kDa in P. e2icJ.i. t um.
These results suggest the monomeric nature of the parasite
haemoglobin and that G. crumeni^X§£ haemoglobins consist
of either one chain which aggregates to a dimer or two
different chains, only one of which aggregates to a dimer
or the two chains may be distinct chemical species with
one chain exhibiting a propensity towards dimerizatIon.
^' §2l£iiiH!5 haemoglobin appears to consist of only one
chain. The SDS-PAGE patterns of G. crumeni^XSI ^"^ P-
f2i£iii!i!!! haemoglobin show that they consist of only one
band having a mobility similar to each other but quite
different from that of the host pigment suggesting that
the parasites and host haemoglobin are chemically distinct
ent i ties.
In the present investigation also, the purified
haemoglobin of J[sogarorchJ^s hyg^e^lobagri^ shows an SDS-PAGE
pattern, clearly different from the fish host haemoglobin.
The fish haemoglobin subunit band was found to have a
molecular weight of about 14 kDa. However molecular mass
estimated by gel filtration is 32 kDa. • This annr.^y is
probably due to the tendency of host haemoglobin to
-52-
dissociate during the process of gel filtration. The second
fraction of the purified parasite haemoglobin appears as
a single band having a molecular mass of about 15 kDa which
is almost consistent with the molecular mass estimated by
gel filtration, suggesting the monomeric nature of parasite
haemoglobin.
The SDS-PAGE pattern of fraction 1 of parasite
haemoglobin along with its low A412/A280 nm ratio indicates
it to bea protein impurity and not haemoglobin.
This information enables us to assume that
i§°E§.£2££lll§ ^ZES5l°^£8.£i possesses a monomeric form of
haemoglobin which is different from host haemoglobin in
terms of molecular weight and eleetrophoretic mobility in
simple and SDS-PAGE systems, as well as by gel filtration
chromatography in Sephadex G-75.
Further studies involving still stringent experiments
such as isoelectric focusing, two dimensional gel
electrophoresis. peptide mapping, amino acid sequence analysis
X-ray crystallography and oxygen affinity are to be carried
out and some of them are in progress to establish the nature
of trematode haemoglobin at the level of primary, secondary
and tertiary structure and its functional aspects.
REFERENCES
Andrews, P. (1964). Estimation of molecular weights of proteins by Sephadex gel filtration. Biochem. J., 91: 222-223.
Barrett, J. (1981). Biochemistry of parasitic helminths. Macmillan, London, pp 49-54.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem^ 72: 248-254.
Braunitzer, G. (1958). The primary structure of the protein components of several haemoglobins. Z. Physiol. Chem., 312: 72-84.
Cain, G.D. (1969a). Studies on haemoglobins in some dlgenetic trematodes. J. Parasltol., 55: 301-306.
Cain, G.D. (1969b). Purification and properties of haemoglobin from Fasclolopsis buski, J. Parasitol., 55: 311-320.
Chapman, C.G. a Mitchell, G.F. (1982). Proteolytic cleavage of immunoglobulin by enzymes released by Fascio la hepatica. Vet. Parasitol., 11: 165-178.
Davenport, H.E. (1949). Proc. Roy. Soc. London, Ser.B, 136: 255. The hemoglobin of Ascaris perienteric fluid. I. Purification and spectra. Blochim. Blophya. Acta, (1965) 111: 485-495.
Fischer, H. Q Zeile, K. (1929). Synthesis of haematoporphyrin, protoporhyrin and hemln. Ann. Chem., 468: 98-116.
Florkin, M. (1969). Respiratory proteins and oxygen transport. In Chemical Zoology (Edited by Florkin M. Q Sheer B.), Vol. 4: 111-134, Academic press, New York.
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