CHAPTER III

BIOCHEMICAL STUDIES
 49

Primary identification of kinetoplastid flagellate is normally based on

morphological character although recently a large variety of new
biochemical methods have been developed and implemented. These
biochemical studies have indicated clearly that the morphological changes
are related in many instances with biochemical changes. Such changes have
been related to the adaptation of the parasite to its hosts.

Until recently, enzyme electrophoresis is the only biochemical

technique extensively used to differentiate and characterize parasitic
Protozoa. Enzymes are important markers as they are stable over many
generations. Enzymes are of particular value in basic genetic studies,
because they are the products of genes, and most of them are under the
control of nuclear genes which undergo a straight forward Mendel ian
pattern of inheritance. Hence enzymes can provide basic information on the
organisation of the parasite genome. Enzyme forms are also remarkable for
their stability over many generations.

In the present chapter the following biochemical parameters were

used for characterizing B. indica n.sp., H. indica n.sp. and L. donovani.

1. Estimation of protein

2. Estimation of carbohydrate

3. Molecular weight determination by sodium-dodecyl-sulphate-polyacry-

lamide gel electrophoresis.

4. Enzyme electrophoresis.

3.1. MATERIALS AND METHODS

3.1.1. Estimation of protein by Folin-Ciocalteu's phenol reagent

The protein content of the antigens was estimated according to the

method described by Lowry et_ a]_. (1951). Bovine serum albumin (BSA,
Sigma) was used as a standard.

Procedure

Standard curve was made using 20 Ug to 10 Ug BSA as on increasing

order of 20, 40 and so on. Each tube was shaked properly with protein
reagent and distilled water to make the final volume upto 1 ml and was
 50

kept in room temperature for 10 minutes. Folin reagent (0.2 ml) was added
to each tube and was incubated at 37°C for 10 minutes, then the O.D.
value was taken at 625 nm in Spectronic-20 (Bausch and Lomb, USA) using
blank for zero adjustment. Data were plotted to obtain a standard curve.
O
The test samples (1x10 organisms/ml) assayed using the above procedure
and the protein content was determined from the standard curve.

3.1.2. Estimation of carbohydrate by phenol sulphuric acid reaction

A rapid and reproducible procedure for the determination of simple
sugars and their derivatives was done according to the method described
by Dubois et_ al_. (1956). Phenol was used as the specific organic colour
developing agent and dextrose (Sigma) was used to the standard sugar for
such determination.

Procedure
In 2 ml aqueous solution (distilled water) containing 10 to 70 yg of
sugar, 0.05 ml of phenol reagent was added. Following this 5 ml of
concentrated sulphuric acid (f^SO^, specific gravity 1.84) was added
rapidly. The mixture was allowed to stand at room temperature for 30
minutes. The colour developed was measured at 480 to 490 nm in Spectronic
-20 (Bausch and Lomb USA) against a blank containing distilled water only.
O
The data was plotted to obtain a standard curve. The test samples (1x10
organisms/ml) were assayed using the above procedure and carbohydrate
(sugar) content was determined from the standard curve.

3.1.3. Molecular weight determination by SDS-PAGE

SDS-PAGE was performed as described by Laemmli (1970). Vertical
slab gel electrophoresis (Bio-Rad Laboratories), was performed using the
SDS-continuous buffer system. Three per cent stacking gel and 5-20%
continuous gradient gel was prepared (by Pharmacia LKB gradient former)
for separation. The composition of gel are shown in table 3.

3.1.3.1. Standard protein

Known standard marker proteins (molecular weight standard kit.
Sigma) consisting of myosin (205 kD), B galactosidase, (116 kD), phospho-
Table 3. The composition of Gel

Constituents 20% 5% 3%

30% polyacrylamide + 5.0 ml 1.25 ml 0.6 ml

0.8% Bis acrylamide

1.5 M Tris-HCI (pH 8.6) 1.5 ml 1.5 ml -

1.5 M Tris-HCI (pH 6.8) - - 0.6 ml

10% SDS 0.075 0.075 0.06 ml

Distilled water 0.9 ml 4.65 ml 4.7 ml

TEMED 2-3 yl 3-4 yl 4 yl

Ammonium per sulphate 30 yl 50 yl 120 yl

(50 mg/ml )
 51

rylase b(97.4 kD), albumin bovine (66 kD), albumin egg (45 kD) and
carbonic anhydrase (20 kD) were used in this study.

3.1.3.2. Preparation of samples

One hundred microgram of soluble protein of all the three organisms

were mixed with sample buffer and boiled in a boiling water bath for 5-10
minutes. In case of standard proteins the boiling was done for 3 minutes.
The samples (both test sample and standard protein) were allowed to cool
down to room temperature and added into the representative wells of
stacking gel.

3.1.3.3. Electrophoresis
Electrophoresis was carried out at 15 mAmp constant current at 15°C
until the samples ran into the stacking gel, the current was raised to 25
mAmp. Electrophoresis was stopped when the dye front migrated to 1-2 cm
of the bottom of the gel.

3.1.3.4. Staining and destaining of the gel

Following electrophoresis the gel was immediately removed from the
glass plate and was transferred to 15% (w/v) Trichloroacetic acid (TCA)
solution for fixation. Fixation was done for 1 hour. Then the gel was
stained with staining solution, for one hour and then destained with
destaining solution for 2-3 days.

3.1.3.5. Molecular weight determination

Subunit molecular weight of the test material were determined by
comparing their electrophoretic mobilities with those standard proteins.
For this the distance of protein migrated and the distance of dye migrated
from the origin of the separating gel were measured. The mobility value
(Rf) was calculated using the following formula :

Mobility (Rf) Distance of protein migration Gel length before staining

Distance of dye migration Gel length after staining

A standard graph was plotted using mobility value of standard

proteins i.e., myosin (205 kD), 3 galactosidase (116 kD), phosphorylase
b (97.4 kD), albumin bovine (68 kD), albumin egg (45 kD), carbonic
 52

anhydrase (29 kD). The molecular weight of unknown protein was calculated
from the standard graph.

3.1.4. Enzyme electrophoresis

3.1.4.1. Organisms
Both L. donovani and H. indica n.sp. were grown in supplemented
defined tissue culture medium (Kar et^ £l_., 1990) for homogenous culture.
Bodo indica n.sp. along with bacteria and bacteria alone were grown in
modified glycerol beef extract medium.

3.1.4.2. Preparation of cell extracts

7
The cloned organisms at log phase (1x10 /ml) were harvested by
centrifugation at 3000 g for 15 minutes at 4°C. The pellets were washed
thrice with phosphate-buffered-sal ine-glucose (pH 8.0) (Lanham and
Godfrey, 1970); resuspended in a hypotonic solution of 1 mM EDTA, 1 mM
dithiothreitol and 1 mM a - aminocaproic acid. The cell lysates were
obtained by freezing (-70°C) and thawing (4°C) the mixtures for six
successive cycles. These lysates were centrifuged at 38,000 g for 30
minutes at 4°C. The supernatants were collected and stored at liquid
nitrogen (—196°C) in the form of 10 Ul beads until required for
electrophoresis.

3.1.4.3. Enzymes tested

The enzymes which have been tested in this study are shown in
table 4. In the present study electrophoretic mobilities of L. donovani, H.
indica n.sp., B. indica n.sp. with bacteria and bacteria alone have been
compared in 11 enzyme system.

3.1.4.4. Electrophoresis
Polyacrylamide gel electrophoresis (5%) was done in Multiphor II
horizontal electrophoresis unit (LKB). An aliquot of 8 Ul was taken from
the specimen sample with a micro syringe and transferred to gel slot. The
electrophoretic conditions and staining components are shown in table 5. At
least 2 tests were made for every enzyme in each culture. Control test
was run in parallel with the experimental tests, but developed without the
specific enzyme substrate. The gel was then fixed in alcohol gel wash or
glycerine and water.
Table 4. Enzyme that were studied

Enzyme Enzyme Enzyme

commission abbreviation
number

Acid phosphatase 3.1.3.2 ACP

Alcohol dehydrogenase 1.1.1.1 ADH

Alkaline phosphatase 3.1.3.1 ALP

Fumerase 4.2.1.2 -

Glucose-6-phosphate dehydrogenase 1 .1.1.49 G6PDH

Glucose-phosphate isomerase 5.3.1.9 GPI

Isocitrate dehydrogenase 1.1.1.42 ICDH

Malate dehydrogenase 1.1 .1 .37 MDH

Malic enzyme 1.1.1.40 ME

Phosphoglucomutase 2.7.5.1 PGM

6-Phosphoglneonate dehydrogenase 1.1.1.44 6PGD

 Table 5. Electrophoretic conditions and developer for the enzyme studied

Enzyme Tank Gel Water Developer Coenzyme Activator Substrate NBT/ PMS Fast
buffer dilution MTT blue

ACP 2 1 15 - B 60ml - 7N NH.OH Phenolphthalate - - Fast

4
monophosphate - 75 mg Garnet
50 mg
ADH 2 1 15 41 ml A 7ml NAD-25mg - 9556 butanol - 2 ml 15mg 1mg
ALP 3 1 15 - B 60ml " Mgcl2-2mg Napthyl acid -
- RR salt
Mncl,,-4mg phosphate - 100 mg 75 mg

Fumerase 2 1 15 40 ml A 10ml NAD-40mg ' Fumaric acid 15mg 1mg ”

(disodium salt) 385 ml
Mai ate dehydrogenase -
- 600 units
*G6PDH 1 1 10 40 ml A 10ml NADP-15mg Mgcl,,-50mg G1ucose-6-phosphate 15mg 1mg -
(disodiura salt) 200 ml

♦GPI 1 1 10 40 ml A 25ml NADP-10mg Mgcl2-40mg Fructose-6-phosphate 15mg 1mg -

(sodium salt)
80mg, Glucose-6-phosphate
dehydrogenase - units
*ICDH 1 1 10 32 ml A 10ml NADP-15mg Mgcl2-50mg Sodium isocitrate 15mg 1mg -
solution (0.1 M) - 8 ml
*MDH 1 1 10 25 ml A 10ml NAD-25mg - Na - L - malate 15mg 1mg -
5 ml (1 M)
ME 2 1 15 35 ml A 10ml KADP-15mg Mgcl^-SOmg Na - L - malate - 5ml(1M) 15mg 1mg -
♦PGM 1 1 10 40 ml A 10ml NADP-15mg Mgcl,,-50mg Glucose-1-phosphate 20mg 1mg
G - 1 - P - 300 mg
G-6-PDH-80 units
G1 ucose-6-phosphate
dehydrogenase
♦6PGD 1 1 10 40 ml A 10ml NADP-15mg Mgcl2-50mg 6-phosphoglucomc acid 15mg 1mg “
(trisodium salt) - 100 mg

*20mg NADP was added to gel mixture

A. Q.2M Tris/HCl {pH-8.0)
B. 0.07M Tr1s/0.005H Citrate (pH-7.0)
1. 0.5M Tr1s/0.016H versene/Q.65M Borate (pH-8.0)
2. 0.13H Tns/0.043M citrate (pH-7.0)
3. 0.15H Citrate/0.24M NaH2P04 (pH-6.3)
 53

3.1.5. Similarity index

For the direct comparison in the pairs of stock, similarity index

was calculated using the following formula:

No. of bands common to both zymodemes

Similarity index
Total No. of bands in both zymodemes

i .e. S 100
x+y

where S = similarity index, x = No. of bands in zymodeme x,

y = No. of bands in zymodeme y, and z = No. of enzyme bands
common to zymodemes X and Y (Gibson et^ al_., 1980).

The similarity index was expressed as the total number of enzyme

bands (for the designation of bands) common to two zymodemes, and
expressed as a percentage. Where bands were absent for enzymes of stock,
the corresponding bands for that enzyme in the other stock of the pair
were not used for the calculation of similarity indices.

3.2. RESULTS

3.2.1. Estimation of protein and carbohydrate

The protein and carbohydrate ratio in B. indica n.sp., H. indica

n.sp. and L. donovani were analysed and it was found to be approximately
3:5:4 and 5:2:4 respectively.

3.2.2. Sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis

The major common polypeptides in SDS-PAGE were observed in the

range of molecular weight at 68 kD, 60 kD, 43 kD, 33 kD and 20 kD
(Figure 40). However, uncommon protein bands were also noticed with
individual parasitic antigen. Herpetomonas indica n.sp. showed higher
molecular weight polypeptide band, then Leishmania followed by Bodo. In
H. indica n.sp. coomassie blue stained polypeptides were seen throughout
the gel ranging from ~ 10 kD to 116 kD. However, in l_. donovani major
polypeptides were observed in the region of ~ 10 kD to 15 kD, ~ 22 kD
to 35 kD and ~ 40 kD to 94 kD. Bodo indica n.sp. showed protein bands
 2 05 kD

1 1 6 kD
97 kD

66 kD

45 kD

29 kD

d c b a

Figure 40 .Comparison of SDS-PACE analysis of protein content of Herpeto-

monas indica n.sp., Leishmania donovani and Bodo indica n.sp.
Electrophoresis was performed in a 5 to 20% gradient poly­
acrylamide slab gel in the presence of 2% SDS and the gel was
subsequently stained with coomassie Brilliant blue. Demonstrated
are H. indica n.sp. (lane b), B. indica n.sp. (lane c), L.
donovani (lane d). The lane 'a1- at the left side of the gel
indicates molecular weight markers. The positions of markers
(Sigma) in KD:myosin, 205; 3 galactosidase, 116; phosphorylase
b, 97 4; albumin bovine, 66; albumin egg, 45; carbonic
anhydrase, 29.
 54

in the molecular weight range of ~ 22 kD to 35 kD, ~ 40 kD to 60 kD and

~ 67 kD to 84 kD.

3.2.3. Enzyme electrophoresis

The results of multiple runs of separately isolated extracts of three

organisms are shown in Figures 41-52. Clear distinctions among the genera
could be seen of the 11 enzymes, although only six enzymes namely, ACP,
ALP, G6PDH, GPI, ICDH and ME distinguished all species with complete
clearity under the given electrophoretic conditions.

3.2.3.1. Acid phosphatase

The ACP isoenzyme patterns were different in three organisms. Some
of the bands were not distinct. Leishmania donovani showed two bands
(one prominent and other light). There were certain amount of 'bleeding'
of bands (Figures 41, 52). Herpetomonas and Bodo showed only one band
but at different position.

3.2.3.2. Alcohol dehydrogenase

In L. donovani and H. indica n.sp. no activity was found. Bodo

indica n.sp. had two alcohol dehydrogenase bands (one prominent) (Figures
42, 52).

3.2.3.3. Alkaline phosphatase

In all organisms, differently migrating bands were found. Both L.

donovani and H. indica n.sp. had only one band whereas Bodo had two
bands (Figures 43, 52).

3.2.3.4. Fumerase

Leishmania donovani showed no fumerase activity. Bodo indica n.sp.

and H. indica n.sp. showed only one band with slightly different
mobility. The intensity of band in B. indica n.sp. was prominent (Figures
44, 52).
Figures41 and 42. Photographs showing the zymodeme patterns of acid
phosphatase and alcohol dehydrogenase respectively.
A = L. donovani, B = H^. indica n.sp., C = 13.
indica n.sp., D = bacteria, 0 = Origin. Anode is at
the top of each zymogram.
Figures43 and 44. Photographs showing the zymodeme patterns of
alkaline phosphatase and fumerase respectively. A =
L. donovani , B = H,. indica n.sp., C = B. indica
n.sp., D = bacteria, 0 = Origin. Anode is at the
top of each zymogram
 55

3.2.3.5. Glucose phosphate isomerase

The GP1 band migrations of H. indica n.sp. and B. indica n.sp.

were same but the activity was much more in H. indica n.sp. than B.
indica n.sp. In L. donovani the banding patterns were different. The
bands were distinct and there were certain amount of 'bleeding' or running
together of bands (Figures 45, 52).

3.2.3.6. Glucose 6 phosphate dehydrogenase

Banding patterns of G6PDH were totally different among the three

parasites. Leishmania donovani had only one band. Herpetomonas indica
n.sp. and B. indica n.sp. had four and five bands respectively. Here
bands were not distinct and certain amount of 'bleeding' and running
together of bands were found (Figures 46, 52).

3.2.3.7. Isocitrate dehydrogenase

Leishmania donovani and 14. indica n.sp. had one ICDH band with
slight difference in mobility. Bodo indica n.sp. had two bands. The major
band was identical with the band of L. donovani and 14. indica n.sp.
(Figures 47,52).

3.2.3.8. Malate dehydrogenase

Leishmania donovani and H. indica n.sp. showed MDH activity. In H.

indica n.sp. it was too much. Bodo indica n.sp. did not show any MDH
activity (Figures 48, 52).

3.2.3.9. Malic enzyme

Malic enzyme banding patterns were different within these three

organisms. Ail the organisms (i.e. L. donovani, H. indica n.sp. and B.
indica n.sp.) had five bands but in different position. Bodo indica n.sp.
had one positive charge band. Some of the bands were not distinct and
certain amount of 'bleeding' of band were found (Figures 49, 52).

3.2.3.10. Phosphogluconate dehydrogenase

Another enzyme which had different migrating of bands in all the

Figures45 and 46. Photographs showing the zymodeme patterns of
glucose 6 phosphate dehydrogenase and glucose
phosphate isomerase respectively. A = L. donovani,
B = H. indica n.sp., C = B. i ndica n.sp., D =
bacteria, 0 = Origin. Anode is at the top of each
zymogram.
Figures 47 and 48. Photographs showing the zymodeme patterns of
isocitrate dehydrogenase and malate dehydrogenase
respectively. A = L. donovani, B = H. indica n.sp.,
C = B. indica n.sp., D = bacteria, 0 = Origin.
Anode is at the top of each zymogram.
 56

organisms were 6PGD. The enzyme activity was higher in H. indica n.sp.
followed by L. donovani and lastly B. indica n.sp. (Figures 50, 52).

3.2.3.11. Phosphoglucose mutase

BothL. donovani and H. indica n.sp. had PGM activity.
Herpetomonas had two PGM bands whereas L. donovani had one. Bodo
indica n.sp. did not show any PCM activity (Figures 51, 52).

3.2.4. Similarity index

The results of similarity index are shown in table 6. It was found

that L. donovani showed close similarity indices with H. indica n.sp. and
then with B. indica n.sp., whereas H. indica n.sp. showed close simi­
larity indices with L. donovani then with B. indica n.sp. Bodo indica
n.sp. showed close similarity indiceswith l_. donovani and then with H.
indica n.sp.

3.3. DISCUSSION

The soluble forms of L. donovani, B. indica n.sp. and H. indica

n.sp. contain both carbohydrate and protein. But in L. donovani and B.
indica n.sp. the carbohydrate contents is more than the protein content
whereas in If. indica n.sp. the protein content is more than the
carbohydrate content.

The common polypeptides in SDS-PAGE proteinprofile analysis were

observed at the molecular weight of 68 kD, 60 kD, 43 kD, 33 kD and 20
kD respectively. These results are well correlated with the findings of
Chang and Fong (1983), Pan (1986), Mortara (1989), where they have
shown that H. samuelpessoai, T. cruzi and L. mexicana antigens expressed
a 43 kD protein which co-migrated with skeletal muscle actin. Our protein
profile showed 43 kD common polypeptide in all the three parasites
thereby suggesting the presence of actin like protein. Apart from this, a
68 kD polypeptide has been found common with the 68 kD protein as
discussed in L. mexicana by Chang and Fong (1983).

Electrophoretic isoenzyme patterns have been employed to identify

genetic heterogenity among the isolates of parasitic Protozoa (Miles et al..
Figures 49 and 50. Photographs showing the zymodeme patterns of malic
enzyme and 6 phosphogluconate dehydrogenase
respectively. A = L. donovani, B = IH. indica n.sp.,
C = B. indica n.sp., D = bacteria, 0 = Origin.
Anode is at the top of each zymogram.
 A B C D
Figure 51. Photograph showing the zymodeme patterns of phospho-
glucose mutase. A = L. donovani, B = H. indica n.sp., C =
indica n.sp., D = bacteria, 0 = Origin. Anode is at the
top of each zymogram.
 G6PDH GPI ICDH MDH

D C 8 A D C B A D C B A

ME 6PGD PGM

Figure 52. Diagrammatic representation of the electrophoretic patterns of

11 enzymes obtained from bacteria, Bodo, Herpetomonas and
Leishmania donovani. Arrows indicate the direction of
movement from the origin (0). A = L. donovani, B = Herpe-
tomonas, C = Bodo, D = bacteria .
Table 6. S imilarity indices among Bacteria, Bodo, Herpetomonas and
Leishmania donovani

Bacteria Bodo Herpetomonas

Bodo 0.700

Herpetomonas 0.277 0.330

Leishmania donovani 0.411 0.450 0.567

 57

1980; Sargeant and Williams, 1979; Siciliano and Shaw, 1976). So far, in
the limited number of enzymes explored, all species of trypanosomatids
have shown distinctive isoenzyme patterns (Tait, 1970; Kilgour and
Godfrey, 1977; Miles et^ aL, 1977; Goncalves de Lima et^ aL, 1979;
Goncalves de Lima et_ aL, 1982) and this generalization is also supported
by the present work. Goncalves de Lima et_ aL (1979) studied six
isoenzymes in different genus of the family Trypanosomatidae. Extensive
work has been carried out on the isoenzyme studies of Leishmania and
Trypanosoma however, no published work is reported on the isoenzyme of
bodonine flagellates. In the present study an attempt has been made to
study the zymodemes of L. donovani, Herpetomonas and Bodo.

In the present study six enzymes (viz. ACP, ALP, G6PDH, GPI, ICDH
and ME) were found in distinguishing theseparasites from each other.
This is the first report of ADH, fumerase and ICDH used in differentiation
of L. donovani, Herpetomonas and Bodo. In MDH enzyme analysis L.
donovani showed two bands, one lightly stained band as also reported by
Kreutzer (1980). Acid phosphatase activity in the present study well
correlate with the findings of Kreutzer (1980) who demonstrated two ACP
bands, one band towards cathodal and other towards anodal ends in L.
donovani. No ALP activity was found in L. donovani by Kreutzer (1980). In
contrast the activity of the same is demonstrated in all three present
isolates. Goncalves de Lima et^ aL (1979) reported only one band of ME in
H. samuelpessoai and L. donovani. On the contrary, the present study
showed two bands of ME in both Herpetomonas and L. donovani. The
possible reason for the differences in the banding patterns may be due to
strain of different source of origin or host response.

In MDH enzyme analysis L. donovani showed two bands, one lightly

stained band as also reported by Kreutzer (1980).

The genetic interpretation of multiple banding pattern of G6PDH, ALP,

fumerase, ME, ICDH, MDH, GPI and 6PGD either in L. donovani, Herpeto­
monas or Bodo raise the possibility of genetically different clones or
diploid nature of the isolates with isoenzyme specified by different alleles
at the same locus. As all the organisms were cloned, the probability of
genetically different clones in the organisms (parasites) were ruled out
(Meloni et aL, 1988). The multiple banded pattern of some enzymes
 58

observed in L. donovani, Herpetomonas or Bodo could explain it as a

heterozygous organism in which the enzyme might be coded by different
alleles at the same locus.

The present results indicate that the isoenzyme electrophoresis is

most significant in kinetoplastid flagellates distinction. The identical
results found in three organisms grown in different media (defined and
complex), indicate none of the isoenzymes studied is repressed or
activated through the nutritional variations in the media. Most of the
differences seen in the enzymes studied here probably represent the
neutral variety, i.e., the molecules have different electrophoretic
migrations but the otherwise kinetically identical or highly similar.

It is clear from this study that the electrophoretic variation occurs

in kinetoplastid flagellates, which serves as convenient marker for genetic
studies in these organisms. The electrophoretic comparison of homologous
proteins offers a quantitative approach to measuring genetic relatedness
(Stout and Shaw, 1973). The present results clearly indicate that
zymography can be useful in delimiting kinetoplastid flagellates. Further,
it can provide insight into the nature of biochemical and genetic
differences. Compilation of electrophoretic profiles of isoenzyme in
kinetoplastid flagellates might provide light on phylogenetic relationship
of order Kinetoplastida. Moreover, comparison of kinetoplastid isoenzyme
profiles with that from Leishmania might point to better selections of
kinetoplastid flagellates as models for expeditious study of the pathogenic
flagellates like Leishmania.