MUTANDWA FELISTERS E

R195689P

COMPARATIVE AND ANIMAL PHYSIOLOGY

PRAC WRITE UP 2

Title: Examining the absorption spectra and testing for copper and iron from the respiratory
pigments in the blood of pulmonate snails.

ABSTRACT

This experiment was conducted so as to examine the absorption spectra and testing for copper and
iron from the respiratory pigments in the blood of plumonate snails. The characteristics of the
respiratory pigments produced by the terrestrial garden snail Helix aspersa and the aquatic planorbid
snail Helisoma trivolvis were also studied. A tiny hole on the shell's dorsal surface was made at the
spire's tip with a dissecting needle and precautions were observed by drying the snail's shell to prevent
water or mucus from contaminating blood samples and not to damage the digestive gland that are
located below the opening. Using a Pasteur pipette, 0.5 cm of blood from each specie was collect into
a clean test tube and diluted with distilled water to a depth of 7 cm. To be sure the blood is completely
oxygenated it was bubbled with oxygen for 30 seconds using a rubber football bladder. The
absorption spectra in the 325–600 nm region against distilled water blanks was measured in
increments of 25nm, then the readings were recorded. The iron and copper testing was also done
using the half of the blood sample in a crucible samples. The samples were added fusion mixture
(equal quantities of anhydrous Na 2CO3 and K2CO3 together) in both the iron and copper testing test
tubes and were heated until organic matter oxidized. A few drops of hydrochloric acid were added
then pH testing was done and 10% ammonium thiocyanate was added in iron testing then in copper,
about 500mg of solid sodium acetate, a drop of pyridine and 1 drop of 10% potassium thiocyanate
solution was added mixed well then decanted the supernatant into a clean test tube then a colour
changes were observed.

INTRONDUCTION

A respiratory pigment is a metalloproteinase that serves a variety of important functions, its main
being oxygen transport. Other functions performed include oxygen storage, carbon dioxide transport,
and transportation of nutrients and gases to cells, and ferries away waste products. There are four
major classifications of respiratory pigment, haemoglobin, hemocyanin, chlorocruorin, and
hemerythrin. The hemecontaining globin is the most commonly occurring respiratory pigment,
occurring in at least 9 different phyla of animals, Prosser, C. L. et al 1952.

Hemocyanin is a respiratory pigment found only in some invertebrates - molluscs (e.g. snails,
octopuses, squids) and arthropods (e.g. scorpions, spiders, horseshoe crabs, lobsters). It occurs in two
forms— oxidized and reduced forms, and in reduced forms the prism shaped or needle-shaped
crystals are soluble in water. The oxygen carrying capacity of hemocyanin is lesser than haemoglobin
Van Aardt, W.B.F., 1981. It is an extracellular protein, i.e. it is not carried in blood cells, but it floats
freely dissolved in the hemolymph. Hemocyanin is a copper-containing protein which gives
hemolymph a blue colour when oxygenated, yet is colourless when deoxygenated, Dilly, P.N.,
Messenger, J.B and Z. Zellforsch.132, 193–201 (1972). The oxygen-binding site is composed of a
pair of copper cations (oxidation state +I when deoxygenated, +II upon oxygenation) which are
directly coordinated by imidazole rings of 6 histidines. Three-dimensional structures of molluscan and
arthropod hemocyanins are different Van Aardt, W.B.F., 1981. The hemocyanin molecules found in
the blood of arthropods are built as hexamers or multiples of hexamers, i.e. subunits depending on the
species in which they are found, each organized into three domains containing five to six α-helices,
domain II contains a four α-helix bundle and encompasses the di-copper centre and domain III is a
seven stranded antiparallel β-barrel. In contrast, molluscan hemocyanins form hollow cylindrical
decamers or multi decamers, Drexel, R. 1987. Their polypeptide chains are large and each consists of
seven or eight functional units, each with a di-copper centre. The structure of the functional unit is
commonly composed of two domains: the α- or core domain consisting of a four α-helix bundle that
houses the di-copper centre and the smaller β-domain incorporating a seven stranded antiparallel β-
barrel.

A haemoglobin (Hb) molecule is a conjugated protein, because it consists of a simple protein and with
a non-protein part. The non-protein part is called prosthetic group. The protein part of the
haemoglobin is called globin (96%) and a porphyrin ring with an iron atom at its centre, called haem
or haematin (4%), Redmond, J.R., 1964. The globin part consists of 4 polypeptide chains-two alpha
(α) chains and two beta (β) chains, Van Aardt, W.B.F., 1981.The globin part helps to prevent oxygen
from binding tightly to haem; when globin is present, oxygen binds reversibly to haem and can be
released to the tissues, Redmond, J.R., 1964. In respiratory organs the haemoglobin combines with
oxygen which form oxyhaemoglobin at normal temperature and pressures.At the low pressure the
oxyhaemoglobin dissociates as oxygen and haemoglobin (HbO2 ↔ Hb + O2)- Haemoglobin is
involved in vertebrates in the transport of respiratory carbon dioxide (about 10% of the total) as
carbamino-haemoglobin in which carbon dioxide is bound to the globin protein, Dilly, P.N.,
Messenger, J.B and Z. Zellforsch.132, 193–201 (1972). The molecular weight of a haemoglobin mol-
ecule is 64,500 daltons. The oxygenated form of haemoglobin is scarlet and deoxygenated form is
bluish-red Redmond, J.R., 1964. The haemoglobin is present in the erythrocytes in almost all
vertebrates except a few Antarctic fish. In the invertebrates they are found in the plasma, coelomic
fluid and haemoglobin-containing cells. In Annelida, the pigment is found in polychaeta (different
kinds of respiratory pigments).In Mollusca the haemoglobin is found in the plasma of Gastropods, and
in Bivalvia, Drexel, R.,. In Chiton and in some prosobranchs the haemoglobin is present in the
muscles of radula as Myoglobin. In Crustacea the pigment is present in small-sized animals (e.g.,
Artemia, Daphnia, and Triops), Prosser, C. L. et al 1952.

Chlorocruorin it is a green coloured respiratory pigment containing iron found in the plasma of some
polychaetes (Serpulid, spirorbid, sabellid fanworms) , Prosser, C. L. et al 1952. It is also found in oxy-
genated and reduced forms. The metalloproteinase of chlorocruorin is similar to haemoglobin except
one vinyl group (CH2 = CH—) is replaced by formyl (HCO—) group Van Aardt, W.B.F., 1981. The
mole weight is 30,00,000 daltons and generally it functions as oxygen carrier, Dilly, P.N., Messenger,
J.B and Z. Zellforsch.132, 193–201 (1972).

Haemerythrin it is an iron containing respiratory pigment found in the blood corpuscles of some
invertebrates (e.g., Sipunculans, Priapulids and inarticulate Brachiopods), Prosser, C. L. et al 1952.. It
is pink or violet coloured in oxygenated state and colourless in deoxygenated state, Van Aardt,
W.B.F., 1981. The mol. weight varies from 40,000 to 108,000 daltons and plays the role of oxygen
storage.A few other less common respiratory pigments are Pinnaglobin—a brown coloured
manganese containing pigment found in the plasma of Pinna (Lamellibranchs).Vanadium—It is a
green coloured vanadium containing pigment found in the vanadocytes of some sea squirts
(Ascidians) Redmond, J.R., 1964.
MATERIALS AND MATERIAL

As per HBZZ409 practical schedule

RESULTS

Part A

Wavelength Distilled water Aquaticsnails Terrestrial snails Helix

Helisoma trivolvis aspersa
325 0 0.453 0.727
350 0 0.433 0.679
375 0 0.506 0.472
400 0 1.731 0.381
425 0 0.969 0.291
450 0 0.205 0.171
475 0 0.082 0.153
500 0 0.058 1.121
525 0 0.208 0.053
550 0 0.138 0.039
575 0 0.094 0.025
600 0 0.011 0.046

Figure1a: shows the table of absorption spectra of aquatic and terrestrial snail’s respiratory pigments.

2 ABSORBANCE SPECTRA
1.8

1.6
1.4
ABSORBANCE m2 mol-1.

1.2

1 Distilled water
0.8 Aquatic snails
Terrestrial snails
0.6

0.4

0.2

0
325 350 375 400 425 450 475 500 525 550 575 600
WAVELENGHT nm

Figure 1b: shows the absorbance spectra of aquatic and terrestrial snails respiratory pigments.

Part B
AQUATIC SNAIL

Iron testing-litmus paper changed from yellow to blue

Copper testing-no colour change

TERRESTRIAL SNAIL

Iron testing-litmus paper changed from yellow to red

DISSCUSION

InLymnaea stagnalis this material has been identified as haemocyanin on a morphological basis.
Histochemical and ultrastructural results on pore cells of 2 snail species(Biomphalaria glabrata,
Planorbarius corneus) having haemoglobin in their blood indicate that the pore cells in these species
synthesize haemoglobin. Possible other functions of pore cells are briefly discussed.

The absorption spectra of haemoglobins from A. glabratus and P. corneus and from B. (P.) globosus
and B. (B.) tropicus angolensis were established. There was no difference between the spectra of
snails which transmit Schistosoma mansoni or S. haematobium and the spectra from related
insusceptible species.The snail haemoglobins produced absorption spectra which differed from
mammalian pigments only in the position of a single peak.Snail blood occasionally produced
additional small peaks in the ultra-violet region, which may have been due to blood proteins other
than the respiratory pigment.

REFERENCE

Drexel, R., Siegmund, S., Schneider, H.J., Lizen, B., Gielens, C., PRÉAUX, G., Lontie, R.,
KELLERMANN, J. and LOTTSPEICH, F., 1987. Complete amino-acid sequence of a functional unit
from a molluscan hemocyanin (Helix pomatia).
Dilly, P.N., Messenger, J.B.: The branchial gland: a site of haemocyanin synthesis inOctopus. Z.
Zellforsch.132, 193–201 (1972).

Targett, G.A.T., 1962. Absorption spectra of blood proteins from intermediate and non-intermediate
hosts of schistosomes. Journal of Helminthology, 36(1-2), pp.201-206.

Targett, G. A. T., 1959.—“Absorption spectra of haemoglobins from intermediate and non-

intermediate hosts of schistosomiasis.” Trans. Roy. Soc. trop. Med. Hyg., 53, 10.

Van Aardt, W.B.F., 1981. Oxygen-binding characteristics of the haemolymph of the freshwater snail
Bulinus (Physopsis) globosus. African Zoology, 16(1), pp.1-4.

Redmond, J.R., 1964. The role of blood pigments in the delivery of oxygen to tissues. Helgoländer
wissenschaftliche Meeresuntersuchungen, 9(1), pp.303-311.

Prosser, C. L., Brown, F. A., Bishop, D. W., Jahn, T. L. and Wulff, V. J., 1952.—Comparative
Animal Physiology. W. B. Saunders Co., Philadelphia and London.