Collagen And Other Macromolecular Composition Of Cuticle Biology Essay

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The objective of this experimental study was to evaluate the collagen and other macromolecular composition of cuticle keeping in view of the usefulness of collagen in various biological applications. Present learning explained by the suggestion that earthworm collagen molecules are fibril kind of filamentous proteins, when observed under light microscope and transmission electron microscope (TEM). On the basis of high magnification studies of TEM, it is revealed that the earthworm cuticle is lacking striations in the gross collagen fibrils. Quantification of collagen present in the cuticular membrane was studied by estimating a marker amino acid, Hydroxyproline by spectrophotometry methods. Apart from the collagen quantification, percent occurrence of other macro-molecules i.e., pentoses, hexoses, and lipid contents were estimated in the cuticular membranes. Based on the results it is concluded that the amount of non-striated collagen present in earthworm cuticles were higher than other invertebrates that the purified collagen can have an advantage in therapeutic and medical applications.

Collagen, a naturally occurring fibrous protein found in humans and other animals providing structural support for bones, skin, tendons, ligaments and blood vessels and is the most abundant protein in the body. Collagen is glycoprotein representing about 40% of the proteins in the adult organism. Seventy five percent of our skin is made up of collagen, providing texture, resiliency and shape; and in total about 30 percent of our body is collagen.

(http://www.entdr.com/collagen.html)

As age progress, the production of collagen slows down. Hence, collagen supplementation is essential to support the body during these natural and at the time of injury or trauma processes. Most people experience that the collagen supplementation is working - including improved sleep, increased energy overall toning, rejuvenation and a greater overall sense wellness. Liquid collagen is the active key ingredient in this collagen product. It helps the body to restore its collagen base by providing highly absorbable collagen protein that nourishes the body. Fibers of collagen are woven together like threads in fabric to form a framework into which new cells can grow. In spite of its critical function, when the body needs to build any new cellular structure, as in the healing process, collagen and collagen fragments play a central role.

It is of huge pharmaceutical interest as many problems of old age are collagen related. Osteoporosis (condition of decreased bone mass) has been recognized as a major public health problem for less than two decades. Pharmacological therapies that effectively minimize the Osteoporosis by introducing an external collagen as a substitute to improve bone mass and are now available widely in countries around the world.

Collagens are still employed in the construction of artificial skin substitutes used in the management of severe burns. These collagens may be bovine or porcine and are used in the combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances.

Recently an alternative to bovine - derived collagen has become available. Although expensive, this recombinant human collagen seems to avoid immune reactions described above for collagen derived from livestock.

The use of cattle as the main sources for collagen has to be reconsidered because of BSE and TSE. Especially the use of BSE as a horrifying inherent threat to collagen products based on the bovine material. The risk of contamination has to be evaluated on a case-by-case basis.

The factors, which have to be considered for bovine collagen, are

The country of origin and herd control; preferably by not choosing certified herds.

The starting material (tendon) is used and was considered of low infectivity but they may be exposed to high infectivity parts during slaughter.

The bovine collagen showing major risks immunologically when they were used in the medical applications.

One alternative is the use of porcine collagen or, much safer collagen from earthworm are eco-balanced by culturing them invitro in order to produce more quantity of collagen for all the required applications which could be an ultimate alternative for avoiding ethical problems evolved by the slaughtering of animals without control in various countries.

1.1 Collagen

1.1.1 Composition and structure

A distinct feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-X-Pro or Gly-X-Hyp, where X may be any of various other amino acid residues. Gly-Pro-Hyp occurs frequently. In collagen, Glycine is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycine's single hydrogen atom. For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids thermally stabilize the triple helix, - Hyp even more so than Pro - and less of them are required in animals such as fish, whose body temperatures are low. Two representations of a collagen helix were given by Goodsell (2006) and collagen has been acknowledged as "Molecule of the Month" for many times (Figure).

Collagen has an unusual amino acid composition and sequence:

Glycine is found at almost every third residue

Proline makes up about 9% of collagen

Two uncommon derivative amino acids not directly inserted during translation of mRNA but found at specific locations relative to glycine and modified post-translationally by different enzymes, both of which require vitamin C as a cofactor.

Hydroxyproline (Hyp), derived by hydroxylation of proline as a post-translational modification.

Hydroxylysine, derived from lysine. Depending on the type of collagen, varying numbers of hydroxylysine have disaccharides attached to them.

Fig. A: Two representations of a collagen helix (David S. Goodsell, 2006).

1.1.2 Types of collagen

Collagen is the major protein comprising the ECM. Collagen occurs in many places throughout the body. There are 28 types of collagen described in literature, of which collagens of 12 types are having clear composition of different chains. Types I, II and III are the most abundant and form fibrils of similar structure. Type IV collagen forms a 2D reticulum and is a major component of the basal lamina.

Type

Chain Composition

Localization

I

[a1(I)]2[a(I)]

Skin, tendon, bone, etc.

II

[a1(II)]3

Cartilage, vitreous humor

III

[a1(III)]3

Skin, muscle, frequently with type I

IV

[a1(IV)2[a2(IV)]

All basal lamina

V

[a1(V)][a2(V)][a3(V)]

Most interstitial tissue, associated with type I

VI

[a1(VI)][a2(VI)][a3(VI)]

Most interstitial tissue, associated with type I

VII

[a1(VII)]3

Epithelia

VIII

[a1(VIII)]3

Some endothelial cells

IX

[a1(IX)][a2(IX)][a3(IX)]

Cartilage, associated with type II

X

[a1(X)]3

Hypertrophic and mineralizing cartilage

XI

[a1(XI)][a2(XI)][a3(XI)]

Cartilage

XII

a1(XII)

Interacts with types I and III

1.1.3 Properties of collagen

Complete amino acid analysis for all collagens are now available to distinguish them from other proteins to characterize collagens as follows:

1. A characteristic wide-angle X-ray-diffraction pattern (the definition of collagen).

2. An infrared-absorption band at 3330 cm­-1.

3. A content of Glycyl residues near to one-third of the total number of residues present.

4. Hydroxyprolyl residues.

5. High contents of Pyrrolidine residues compared with other proteins.

6. Low contents of Cystine, Methionine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine and Histidine.

Characteristics

Vertebrate collagens and fibers from cuvierian tubules of H. forskali

Cuticle collagen of Ascaris and Lumbricus

X-ray diffraction

Meridian low-angle pattern

No low-angle pattern yet observed

Electron microscopy

Fibrils banded in electron micrographs

Fibrils apparently not banded

Taxonomy

Deuterostomia

Protostomia

Chemical features

Hydroxylysyl residues

No Hydroxylysyl residues

About one hydroxylic side chain in every six residues

Ascaris cuticle has one hydroxylic residue in every sixteen residues, and Lumbricus cuticle has one in every three residues

Prolyl and hydroxyprolyl contents of the same order

Prolyl and Hydroxyprolyl contents of different orders

Table A: Characteristic differentiation between the vertebrate collagens and invertebrate collagens (Watson and Silverster., 1959)

1.1.4 Uses of Collagen

In 1970s and the 1980s expanding medical applications of bio materials and connective tissue research challenged academically oriented scientists and commercial research laboratories to focus their studies collagen as it is the chief biomaterial used in various purposes such as cosmetic production, leather processing, pharmaceutical and etc.

The native, basic chemical properties of collagen are necessary to understand the effects of the isolated collagen and the potential modification of these collagen materials are involved in the synthesis and application of biologically important compounds that are used as implantable minipellets loaded with protein drugs, drug carriers, collagen gels and shields in ophthalmology, injectable dispersions for local tumor treatment, antibiotics, film building polymer in cosmetics and tissue grafting sheets, meshes as well as collagen sponges for tissue engineering purposes.

The other uses of collagen

1.1.4.1 Industrial uses

Nutritionally collagen is considered as poor quality protein because they lack adequate amounts of some of the essential amino acids. Some collagen based dietary supplements are claimed to improve skin and fingernail quality and aid joint health.

Collagen is the word derived from the Greek word kola, which means "glue producer". Collagen adhesive was used by Egyptians about 4000 years ago and Native Americans used it in bows about 1500 years ago. The oldest glue in the world carbon dated as more than 8000 years ago, was found to be collagen - used as a protective lining on rope baskets and embroidered fabrics and to hold the utensils together. Animal glues are thermoplastic, softening again upon reheating and so they are still used in making musical instruments such as fine violins and guitars, which may have to be reopened for repairs - an application incompatible with tough, synthetic plastic adhesives, which are permanent. Gelatin-resorcinol-formaldehyde glue has been used to repair experimental incisions in rabbit lungs (Ennker., 1994).

1.1.4.2 Medical uses

Collagen has been widely used in cosmetic surgery and certain skin substitutes for burn patients. The cosmetic use of collagen is declining because:

There is fairly high rate of allergic reactions causing prolonged redness and requiring inconspicuous patch testing prior to cosmetic use.

Most medical collagen is derived from cows, posing the risk of transmitting prion diseases like BSE.

Alternative using the patient's own fat or hyaluronic acid are readily available.

Release of oligosaccharides from glycoproteins (Davies et al., 1994) is also a considering factor in collagen applications.

1.2 Earthworm as a source of collagen

Earthworms belong to the Phylum Annelida and Class Oligochaeta, which consists of over 7000 species. Their bodies are long and tube-like, tapering on both ends and commonly ranging in length from one to six inches. Certain Australian earthworms are several feet long. Another characteristic of the Phylum Annelida is a segmented body, including an enlargement of several segments to produce the clitellum, a glandular organ used for reproduction. Earthworms are hermaphroditic and homosexual, and thus they may function as either a male or a female during reproduction. Self-fertilization does not occur.

Earthworms are cosmopolitan in distribution, i.e., found almost all over the world except the Arctic and Antarctic regions. Indirectly they provide food for man by their beneficial effects on the plant growth. They aerate the soil, promote drainage and draw organic materials into their burrows where it decomposes producing nutritive materials for growing plants. Earthworms also serve as fish bait and the name Angel worm. Earthworms are grown virtually in all soils in which moisture and organic content are rich. Earthworms are found in a wide range of habitats throughout the world, having adapted too many different soil types as well as to lakes and streams. Earthworms are often called night crawlers, garden worms, red worms or, simply, worms; are a valuable resource to many people. They provide bait for fishing, a source of protein for food, and most importantly, they play a unique and important role in conditioning the soil.

With the advent of chemical pest control, however, earthworms have become non-target recipients of many pesticides. Some of the most effective pesticides are broad spectrum in action, and they may inadvertently harm earthworms and other beneficial soil organisms. Harmful substances ingested by earthworms also may be concentrated in the food chain.

Although one acre of soil may hold up to eight million earthworms, most people pay little attention to these productive and beneficial animals. They mostly go unnoticed from day to day, unless a heavy rain forces them to the surface of the soil, an angler needs some bait, or their casts disrupt a game of golf.

Fig. B: Earthworms (Eisenia foetida)

1.2.1 Builders of Soil

Earthworms benefit the soil in many ways, primarily due to the physical and chemical effects of their casts and burrows. Earthworm casts, consisting of waste excreted after feeding, are composed mostly of soil mixed with digested plant residues. Casts modify soil structure by breaking larger structural units (plates and blocks) into finer, spherical granules. An exception to this has been reported from some Canadian clay soils in which, during wet weather, worms can convert a soil structure to a massive paste. As plant material and soil passes through an earthworm's digestive system, its gizzard breaks down the particles into smaller fragments. These fragments, once excreted, are further decomposed by other worms and microorganisms. Earthworm casts can contribute up to 50 percent of the soil aggregates in some soils.

Cast production is most abundant in moist spring and fall seasons when earthworms inhabit surface layers of the soil. During this time, 20 casts per square foot of soil surface are not uncommon, and as much as 40 pounds of casts per 1000 square feet per year have been recorded. Under conditions of extreme temperatures or moisture stress during summer and winter, earthworms migrate downward into subsoil horizons, and enter a resting state called aestivation. In irrigated areas, such as golf course greens, fairways, and tees, this behavior may be altered and earthworms may not descend during the summer months. Thus, their activity may be regarded as a problem requiring management.

1.2.2 Cuticle of Earthworms

The cuticle, is easily examined by various physical and chemical method, was analyzed minutely by Singleton (1957), Maser and Rice (1962), who found 80% of it are proteins (with a high content of Hydroxyproline, Glycine and Alanine), and 20% are carbohydrates. When soluble earthworm cuticle collagen molecules are subjected to the shearing forces of a flow birefringence instrument, they are broken into particles approximately half the original size (Maser., 1963). Along with these results, the X-ray diffraction studies also pointed out the presence of the collagen. It is thought that the collagen molecule of Oligochaeta is twice the weight and the length of that of the vertebrates (Baccetti., 1967). It was Ruska (1961), who visualized in the electron microscope a 560°A cross binding in the fibrils of the sub epidermal layer of Lumbricus, and Van Gansen (1962) observed fibrils with periodicity, 140 mµ in Eisenia. In earthworms, the external and internal collagens seem to differ in structure (Watson., 1958., 1959). ACL of rabbit shows fine fibrillar collagens under electron and light microscopy as narrated by Bayat et al., 2003 where these structures almost having morphological similarity with that of earthworm collagens.

The maintenance of earthworms and the invitro culture of these invertebrates may give data prior to the biotechnological applications of cuticular constituents and also to analyze agricultural significance of these macro molecules. Present evaluations can be used as base data for the followers to look in to molecular or biotechnological aspects of collagen and other cuticular components for the benefit of mankind.

2. Objective and scope of work

Collagen obtained form different animals, particularly from bovine origin is used in many medical and pharmaceutical applications including tissue engineering and drug delivery materials (Friess., 1998; Elsner., 1994) due to its strength and stability as well as its general compatibility with human tissues. The safety images of the collagen originated from the animals have been getting down due to existence of heavy metals in their body in huge concentration.

The main focus of the proposed work is to avoid such problems and to isolate collagen from lower vertebrates. As the evolution goes on, the vertebrate source of collagens, despite of their vast application, showing high immunogenicity that leads to secondary complications to the consumer. The lower organisms like earthworms and the other marine organisms like Sponges, Molluscans and Echinoderms are less complex in their body make and participated actively in the race to produce safer collagens to meet the biocompatibility practices with Human physiological systems. Because of its simplicity of isolating collagens from cuticles of this creature, it has become an effective tool for medical researchers trying to unravel the workings of the human immune system. Hence in the present study earthworms are chosen for the isolation of the collagen.

3. Materials and methods

All chemicals and reagents used in the present study were of analytical grade and were used without any further purification.

3.1 Test Organism

Earthworms of specific variety are used for different purpose. Borrowing type of worms with their strong muscles is capable of borrowing as deep as three meters and thus protects themselves from drought conditions. Exotic species like Eisenia foetida, Edurilus euginea, Perionyx erobicola, Decogastor bolavil, Dravida witsiu, Lampitto mauriti are useful for biochemical and biotechnological applications and for the research work. They can tolerate at high temperature and found in organic rich soil environment. The two types of earthworm best suited to worm composting or vermibiotechnology are the red worms: Eisenia foetida (commonly known as red wiggler, brandling, or manure worm) and Lumbricus rubellus, where Eisenia was chosen in this present study due to its cosmopolitan availability.

Eisenia foetida (Savigny):

These earthworms reproduce sexually and releases up to 900 eggs per year per worm. The period of life cycle of Eisenia foetida is 78 days and mature at 50th day of its birth. They start producing cocoons on 55th day, which take incubation period of 23 days and release 1-9 hatchings. These are present in less number on dry soils. The cuticle, which is resistant to a variety of enzymes, is composed of non-striated, bundles of probable collagen fibers that are orthogonally oriented and are embedded in a proteoglycan matrix (Burke., 2005). Adult earthworms have the capacity of producing more amount of collagen in cuticles. Hence the earthworms are chosen for isolation of cuticle.

3.1.1 CLASSIFICATION AND TAXONOMIC POSITION

Kingdom

:

Eukaryota

Phylum

:

Annelida

Super class

:

Clitellata

Class

:

Oligochaeta

Order

:

Haplotaxidae

Sub order

:

Lumbricina

Super family

:

Lumbricoidae

Family

:

Lumbricidae

Genus

:

Eisenia

Species

:

Foetida

The earthworms, Eisenia foetida, popularly known as tiger worm or European worm, obtained from the vermiculture project, Kothapet fruit market, Dilsukhnagar, Hyderabad, Andhra Pradesh in India. They were carefully brought to laboratory along with moist soil in porous cotton bags. They were grown under the laboratory conditions using artificial soil method according to the OECD (Organization for Economic Co-operation and Development) guidelines. The composition of artificial soil method is 70% of sand, 20% clay, and 10% peat or cow dung.

Fig. C: Earthworm (Eisenia foetida)

3.1.2 Acclimatization and breeding of the worms

The worms were acclimatized for seven days under the laboratory conditions by releasing them into feed boxes (50 X 50 X 15 cm) containing 50:50 cattle manure and dried Lucerne grass. Wet gunny bags were placed as a cover on the feed boxes. The same media was also used for breeding the worms at laboratory conditions with 20  2°C, pH 7.0, relative humidity 60% and twelve hours light/ dark cycle.

3.2 Isolation methods

Six batches of earthworms were taken from the 7 day acclimatized feed boxes, which were washed thoroughly in two changes of tap water and then immersed in anhydrous diethyl ether on elevated mesh screens in order to allow residual water to settle out. After 15-30 minutes in ether, the worms were removed singly and the cuticle is peeled off with the help of forceps (Maser et al., 1962).

Immediately after the removal, the cuticles were placed in 1ml of 0.5% acetic acid per 10 cuticles and kept for stirring at 4° C for 48 hrs for the extraction of collagens (Miller et al., 1982). After stirring, the cuticle solution was taken from stirrer and is transferred to the centrifuge tubes for low-speed centrifugation in order to remove the insoluble material. The supernatant suspension was re-centrifuged at 18000 rpm for 10 minutes. The pellet was collected and the supernatant was taken for salt-out the protein (2M NaCl). The pellet is then immersed in 0.5 M Acetic Acid (pH - 3.0) with the protease inhibitors cocktail (30 µl) (1 mM PMSF, 2 mM NEM, and 1µg/ml pepstatin). After adding the protease inhibitors cocktail, the solution was sonicated and kept stirring for 12-24 hrs at 4° C. After stirring, the solution was filtered through nylon cloth, the remaining residue on nylon cloth was washed with water containing all the inhibitors and the process was repeated if the residue was sufficient. To the filtrate, pepsin (1:10 w/w) was added to the dry weight of the cuticle and kept for stirring (slow stirring) at 4°C overnight. The solution was centrifuged at 35000 x g for 1hr at 4° C. Measure the volume of the supernatant and 2 M NaCl was added to the solution; again the solution was stirred for 1 hr at 4° C. Spin the solution at 50000 x g for 2 hrs. The pellet was taken and dissolved in 0.05 M ascetic acid. Thus, the protein was isolated by using the protease inhibitors. The isolated cuticle collagen was proved by protein estimation (Bradford method) and Hydroxyproline (marker imino acid of collagen) estimation. It is characterized by SDS-PAGE and structural observations by LM and TEM.

3.3 CHARACTERIZATION OF COLLAGEN

3.3.1 PROTEIN ESTIMATION (Bradford method)

The Protein was estimated using Bradford Reagent.

Reagents required

Bradford reagent

Bradford reagent was prepared by dissolving 100 mg Coomassie Blue G-250 in 50 ml of 95% Ethanol, add 100 ml of 85% Phosphoric acid and dilute to a one liter. The reagent was filtered at least once and perhaps more, since it seems to precipitate dye on over time.

Assay reagent

The assay reagent was prepared by diluting 1 volume of the dye stock with 4 volumes of distilled H2O.  The solution appeared in brown and had a pH of 1.1.  It is stable for weeks in a dark bottle at 4° C.

PROCEDURE

50 µl of the sample was allowed to react with 250 µl of Bradford reagent in a homogenous condition and allowed to incubate for 30 minutes at room temperature. The absorbance of the sample was read at 595 nm (Red filter) against the blank in UV-Visible Spectrophotometer (Molecular Device, USA; with an advanced software Softmax Pro 3.0). The total protein was estimated.

3.3.2 ESTIMATION OF 4-HYDROXYPROLINE (Jamall method)

The total amount of Hydroxyproline present in collagen is to be estimated.

Chemicals required

Concentrated HCl

Chloramine T

Isopropanol

Reagents required

Ehrlich's reagent (p-dimethylaminobenzaldehyde).

Procedure

100 µl of isolated cuticle collagen sample was taken and was hydrolyzed by adding 100 µl of concentrated HCl for 18 hrs at 105° C. After hydrolysis, 25 µl of sample was taken in separate vials (as triplicate) and allowed to evaporate till the complete dryness under vacuum. 1.2 ml of 50% isopropanol was added to the dried samples and then 0.2 ml of 0.56% Chloramine T solution was added. After 10minutes, 1ml of ER was added to give a final volume of 2.4 ml with a calculated amount of 19.9 mM of Chloramine T. Mix the solution well and incubate the test tubes at 50° C for 90 minutes. Finally samples were allowed to cool and obtained the OD values at 558 nm.

Table 3: OD values of different Hydroxyproline concentrations at 558 nm

3.4 SDS- PAGE ELECTROPHORESIS

The isolated collagen is characterized by running SDS- PAGE with suitable marker proteins.

Chemicals required

Sodium Dodecyl Sulphate (SDS)

Ammonium Per Sulphate (APS)

TEMED

Coomassie Blue R-250

Methanol

Acetic acid

Distilled Water

Tris base

Glycine

Preparation of Reagents

A. Acryl amide/ bis Acryl amide:

29.9 gm of Acryl amide and 0.8 gm of N'N' - bis-methylene - acryl amide were added and was made up to 100 ml with distilled water.

B. 1.5 M Tris- HCl, pH 8.8

27.23 gm of Tris base was taken and mixed with 80 ml of water. pH 8.8 was adjusted with 6 N HCl. Then it was made up to 150 ml with water and stored it at 4° C.

C. 0.5 M Tris-HCl, pH 6.8

6.1 gm of Tris base was taken and mixed with water 80 ml of water. pH 6.8 was adjusted with 6 N HCl. It was made to 100 ml of water and stored at 4° C.

D. 10% SDS

10 gm of SDS was added to 90 ml of the distilled water. It was stirred gently to dissolve. Then it was made to 10 ml.

E. Sample Buffer/SDS Reducing Buffer

3.8 ml of distilled water, 1.0 ml of 0.5 M Tris - HCl (pH 6.8), 0.8 ml of Glycerol, 1.6 ml of 10% SDS, 0.4 ml of 2- mercaptoethanol and 0.4 ml of 1% Bromophenol Blue were added to dilute the sample 1:4 and was heat at 95° C for four minutes.

F. 5 X Running Buffer, pH 8.3

9.0 gm of Tris base, 43.2 gm of Glycine, 3 gm of SDS were added and made up to 600 ml with water. It was stored at 4° C. It was diluted to 60 ml of 5 X buffer with 240 ml of water for one run of the gel.

Procedure

10% resolving gel was prepared by taking 1.8 ml of distilled water, 1.8 ml Acryl amide, 1.3 ml of 1.5 M Tris, 50 µl 10% SDS in a test tube. Then 50 µl of 10% APS and 5 µl of TEMED were added, mixed thoroughly and finally poured into glass plates and it was allowed to solidify. After resolving gel solidification, 5% of stacking gel was prepared by taking 1.95ml of distilled water, 1.7ml of Acryl amide, 1.25ml of 0.5M of Tris, 50µl of 10% SDS in a test tube, then 50µl of APS and 5µl of TEMED added and poured above the resolving gel into the glass plates, then comb was inserted and allowed to solidify. After complete solidification the comb was removed from the glass plates carefully. Sample was mixed with sample buffer and boiled at 95°C for 4 min and allowed to cool. The samples were added into the wells along with suitable marker protein in one well. The electrophoresis was carried out at 70volts till the sample reaches the bottom of the gel. After the completion of electrophoresis process, the gel was immersed in fixative solution (10% ascetic acid, 45% methanol, 45% distilled water), and then gel was washed with distilled water again immersed in staining solution (10% ascetic acid, 45% methanol, 45% distilled water and 0.05% of coomassie blue R-250) for overnight. The gel was kept for distaining in distaining solution (10% ascetic acid, 45% methanol, 45% distilled water) for the removal of the dye. Finally the gel was washed and observed for the presence of the bands by taking photographs with gel documentation unit.

3.5 MICROSCOPIC STUDIES

3.5.1 Preparation for LM

The isolated cuticle collagen smear was taken on to the microscopic glass slide. It was allowed to dry for sometime. After drying, the glass slide was observed using the Light Microscope. When it was observed at higher resolutions the fibrils were clearly observed.

3.5.2 Preparation for TEM

Whole cuticles that had been stored in the cold for 1-14 days in 0.5% acetic acid were prepared for electron microscopy by mechanical dispersion. Drops of the suspensions were placed on the substrate-covered grids, and the excess blotted off. The grids were washed with water and stained with 1% Phosphotungstic acid in acetate buffer (pH 4.2-5.6). Similarly, the isolated collagen grids were prepared and they were observed using the transmission electron microscope.

3.6 ESTIMATION OF MACROMOLECULES

The various macromolecules that are estimated are carbohydrates, lipids.

3.6.1. Carbohydrates Estimation (Dubois et al.1956)

The total carbohydrate content was assayed by the phenol - sulphuric acid method.

Reagents required

(1) Sulfuric acid: Reagent grade 95.5% conforming to ACS specifications, specific gravity 1.84.

(2) Phenol (80%): It was prepared by adding 20 grams of glass distilled water to 80 grams of redistilled reagent grade phenol.

Procedure

2ml of sugar solution was pipetted into a test tube, and 0.05 ml. of 80% phenol was added. Then 5ml of concentrated sulfuric acid was added rapidly, the stream of acid was directed against the liquid surface rather than against the side of the test tube in order to obtain good mixing. The tubes were allowed to stand 10 minutes, and then they were shaken and placed for 10 to 20 minutes in a water bath at 25° to 30°C., before readings were taken. The color was stable for several hours and readings may be made if necessary. The absorbance of the characteristic yellow orange color was measured at 490 nm (green filters) for hexoses and 480 nm (green filters) for pentoses. Blank was prepared by substituting distilled water for the sugar solution. The amount of sugar was determined.

3.6.2 TOTAL LIPIDS (Bligh and Dyer method., 1959)

The total lipid content was estimated by using a 1:2 mixture of chloroform: methanol.

Reagents Required

(1) Chloroform HPLC Grade.

(2) Methanol TLC Grade.

Procedure

In this method, a mixture of chloroform and methanol (2:1v/v) was used. The tissue (about 1g) wet weight was first grinded in a pestle and mortar with about 10 ml distilled water. The pulp was transferred to a conical flask (250ml) and 30 ml of chloroform methanol mixture was added and mixed well. For complete extraction, it was kept overnight at room temperature in the dark. At the end of the period, 20ml chloroform and 20ml water was added. The resulting solution was subjected to centrifugation, where 3 layers were seen. A clear lower layer of chloroform containing all the lipids, a colored aqueous layer of methanol with all water-soluble material and a thick pasty interface were seen.

The methanol layer was discarded and the lower layer was carefully collected free of interphase by sucking out with a fine capillary. The organic layer from either of the extraction method was taken in a pre-weighed beaker or vial and was evaporated carefully. The sample was covered with a dark paper to protect from light in order to avoid polymerization or decomposition of some lipids on exposure to light, heat and oxygen.

When the solution was free of organic solvents, the total lipid content was determined and the results were expressed in terms of weight in µg of total lipid per gm dry tissue.

4. RESULTS

In the present study, isolation of the cuticle collagen was carried out from earthworm Eisenia foetida. The cuticle was peeled from the organism carefully using the fine forceps. A preliminary quantification of the cuticle was done and the process was carried out for the isolation of cuticle collagen. The isolated cuticle collagen was characterized by SDS PAGE, Hydroxyproline estimation. The total protein amount was estimated in isolated cuticle collagen. Biochemical parameters were studied for the presence of the carbohydrates (Pentose and Hexose sugars) and also estimated the amount of lipids present in isolated cuticle collagen.

4.1 Maintenance of the Organism

The earthworms were obtained from the vermiculture project, Kothapet fruit market, Dilshukhnagar, Hyderabad, Andhra Pradesh in India. They were brought to laboratory carefully in moist soil in porous cotton bags and were grown under laboratory conditions using the artificial soil method according to OECD guidelines.

4.2 Peeling of the cuticle

A batch of earthworms were taken and immersed in dry ether (Figure 1&2) and finally the cuticle was removed carefully using the fine forceps (Figure 3).

Figure 1: Normal appearance of the earthworm

Figure 2: Appearance of the earthworm body after dipping in the ether

Figure 3: Removal of the earthworm cuticle

4.3 Quantification of the amount of the cuticle

Before starting the experiment the weight of each earthworm was taken. The net weight of the total earthworms were weighed as 166.581 gm approximately 150 earthworms (mean weight of each worm weighs 1.11  0.04179 gm). After separating the cuticle from the earthworms, the net amount of the isolated cuticle was 8.319 gm. The results clear-cut that 4.97 gm of cuticle can be isolated from 100 gm of adult earthworms (Table 1).

Replicate

No of Earthworms

Weight of Earthworms in gm

Weight of the isolated cuticle in gm

R-1

20

19.3

1.11

R-2

27

29.46

1.49

R-3

35

36.12

1.94

R-4

20

24.2

1.11

R-5

28

33.82

1.55

R-6

20

23.68

1.11

Total

150

166.581

8.319

Mean ± SE

27.76±2.65

1.38±0.14

* The results indicated that precisely 4.97 gm cuticle can be isolated from 100 gm of adult earthworms.

Table 1: Quantification of the cuticle from the earthworms

4.4 Protein content

The protein content present in the sample was estimated by taking the values from the standard graph (graph 1).

From Graph 1:

Linear Regression for Protein estimation Y = A + B x X

Parameter

Value

Error

Intercept (A)

0.02549

0.00994

Slope (B)

0.0832

0.00256

Y

=

A

+

B

x

X

Y

=

0.02549

+

0.0832

x

X

X = (Y - 0.02549) / 0.0832

Unknown sample protein conc. = (Observed OD - 0.02549) / 0.0832

Calculation

The amount of wet cuticle taken was 5.546 gm for the isolation of the proteins. From the sample about 10 µl of protein was taken for protein estimation by using the standard equation we have the regression equation as

Y = A + B x X

Where Y = OD value taken from spectrophotometer = 0.3717

A = X- intercept from the graph (0.0255)

B = slope of the line from graph (0.0832)

X = (Y-0.0255)/ 0.0832

= (0.3717-0.0255) / 0.0832

= 0.416mg of protein/ ml of the purified cuticle sample

Protein Concentration

(µg)

Optical density at 595 nm (Green filter)

Replicate 1

Replicate 2

Replicate 3

Mean ± S.E

0.625

0.066

0.062

0.058

0.062±0.00231

1.25

0.149

0.133

0.112

0.13133±0.01071

1.875

0.186

0.192

0.197

0.19167±0.00318

2.5

0.232

0.234

0.238

0.23467±0.00176

3.125

0.319

0.275

0.29

0.29467±0.01291

3.75

0.334

0.353

0.357

0.348±0.00709

4.375

0.395

0.392

0.389

0.392±0.00173

5

0.44

0.37

0.481

0.43033±0.03241

6.25

0.55

0.553

0.587

0.56333±0.01186

Table 2: OD values obtained at 595 nm for different protein concentrations

Graph 1: Protein standard Graph plotted between the concentration of the protein and the absorbance at 595nm

Result

The total amount of isolated cuticle collagen was approximately 0.416 mg per gm of the cuticle peeled from the earthworms. The total percentage of the cuticle collagen peeled from the cuticle of the earthworms was 41.6%.

4.5 4-Hydroxyproline content

4- Hydroxyproline content present in the sample was estimated by taking the values from the standard graph (graph 2).

From Graph 2:

Linear Regression for Hydroxyproline estimation Y = A + B x X

Parameter

Value

Error

Intercept (A)

-0.008

9.971E-4

Slope (B)

0.008

1.092E-4

Y

=

A

+

B

X

X

Y

=

-0.008

+

0.008

X

X

X = (Y + 0.008) / 0.008

Unknown sample Hydroxyproline conc. = (Observed OD + 0.008) / 0.008

Calculation

About 100 µl of the sample was used for the estimation of Hydroxyproline by using the following formula

Y = A + B x X

Hydroxyproline Concentration

(µg)

Optical density at 558 nm (Green filters)

Replicate-1

Replicate-2

Replicate-3

Mean ± S.E

1

0.003

0.006

0.0045

0.0045 ± 0.0015

2.5

0.01

0.01

0.012

0.0107± 0.001

5

0.037

0.028

0.031

0.032 ± 0.0027

7.5

0.047

0.049

0.055

0.0503 ± 0.0024

10

0.067

0.066

0.076

0.0697±0.0032

12.5

0.09

0.081

0.092

0.0877±0.0034

15

0.101

0.11

0.113

0.108±0.0036

17.5

0.13

0.129

0.133

0.1307±0.0012

20

0.157

0.149

0.15

0.152±0.0025

Table 3: Optical density of Hydroxyproline Concentration at 558nm

Graph 2: Hydroxyproline standard graph

Where Y = OD value taken from spectrophotometer = 0.087

A = X- intercept from the graph (-0.008)

B = slope of the line from graph (0.008)

X = (Y- (-0.008))/ 0.00

= (0.087-(-0.008))/ 0.008

= 11.875 µg of Hydroxyproline/ 100 µl of the sample.

Result:

The total amount of the Hydroxyproline present is 0.214 mg per 1 gm of the isolated earthworm cuticle and the collagen quantified based on a unique feature of hydroxyproline presence (13% of all the amino acids) in collagen.

4.6 Electrophoretic (SDS PAGE) Characterization of cuticle collagen:

Earthworm crude as well as purified proteins were electrophoresed on 12% PAGE gel along with a medium Protein Molecular Weight Marker (PMW-M) for collagen Molecular weight countercheck. A band was observed at 64 KD (figure 4) region, indicating the presence of the purified earthworm collagen, compared with the crude cuticle collagen. According to the available literature, this particular protein band region was the presence of a portion of collagen chain under the same extraction procedure for earthworm collagens.

Figure 4: 12% SDS PAGE gel showing the crude and isolated cuticle collagen

4.7 Microscopic studies

These mechanically sheared and the layers of fibrils were observed under the Light Microscope at 300X and 1400X. The crude cuticles showing the fibrils were clearly seen in the figures 5 & 6.

Some of the purified cuticle collagen samples are taken for observing the fibrillar arrangements of collagens using Transmission Electron Microscopy. The close and tightened fibrils are observed at the resolution of 40000X and 55000X, where the fibrils possess unclear striations. The cuticle collagens were clearly seen in the form of fibrils as shown in figures 7& 8.

Figure 5: Crude form of earthworm cuticle collagen at 300X observed under Light Microscope

Figure 6: Crude form of the earthworm cuticle collagen at 1400X observed under Light Microscope

Figure 7: Purified form of earthworm cuticle collagen at 55000X observed using TEM

Figure 8: Purified form of earthworm cuticle collagen at 40000X observed using TEM

4.8 ESTIMATION OF THE MACROMOLECULES

4.8.1 Estimation of carbohydrate

4.8.1.1 Amount of pentose content

The total content of pentose sugars was calculated by taking the OD value at 480 nm.

From graph 3:

Linear Regression for carbohydrate estimation Y = A + B x X

Parameter

Value

Error

Intercept (A)

0.019

0.01

Slope (B)

0.024

0.654052E-4

Y

=

A

+

B

x

X

Y

=

0.019

+

0.024

x

X

X = (Y - 0.019) / 0.024

Unknown sample carbohydrate conc. = (Observed OD - 0.019) / 0.024

Calculations

The amount of wet cuticle taken is 0.352 gm for the estimation of the carbohydrates. From Pentose standard equation we have the regression equation as

Y = A + B x X

Protein concentration (µg)

Optical density at 480 nm (Blue filter)

Replicate-1

Replicate-2

Replicate-3

Mean ± S.E

0.25

0.021

0.029

0.025

0.025±0.004

0.5

0.04

0.029

0.024

0.031±0.009

1

0.05

0.061

0.058

0.055±0.006

5

0.151

0.083

0.126

0.12±0.37

10

0.156

0.376

0.266

0.266±0.11

25

0.749

0.495

0.498

0.581±0.168

30

0.656

0.853

0.741

0.75±0.359

Table 4: OD values of respective protein concentrations at 480nm (for Pentose sugar estimation)

Graph 3: Ribose sugar standard graph

Where Y= OD value taken from spectrophotometer = 1.254

A = X- intercept from the graph = 0.019

B = slope of the line from graph = 0.024

X = (Y-0.019)/0.024

= (1.254-0.019)/0.024

= 51.45 µg of Pentose sugars/0.352 gm of cuticle

Result:

It was clearly resulted that 146.2 µg of Pentose sugars are present in 1 gm of the isolated cuticle collagen. Approximately 0.146% of the Pentose sugars are present in the isolated cuticle collagen.

4.8.1.2 Hexose estimation

The total content of pentose sugars was calculated by taking the OD value at 490 nm.

From Graph 4:

Linear Regression for Protein estimation Y = A + B x X

Parameter

Value

Error

Intercept (A)

0.004

0.025

Slope (B)

0.041

0.001

Y

=

A

+

B

x

X

Y

=

0.006

+

0.041

x

X

X = (Y - 0.006) / 0.041

Unknown sample protein conc. = (Observed OD - 0.006) / 0.041

Protein Concentration (µg)

Optical density at 490 nm (Blue filter)

Replicate-1

Replicate-2

Replicate-3

Mean ± S.E

1

0.046

0.017

0.024

0.029±0.009

5

0.242

0.246

0.248

0.246±0.006

10

0.494

0.317

0.372

0.394±0.052

15

0.602

0.514

0.577

0.564±0.026

20

0.924

0.754

0.881

0.853±0.051

25

1.086

1.088

1.090

1.088±0.011

30

1.246

1.122

1.221

1.196±0.038

35

1.435

1.438

1.441

1.438±0.015

40

1.612

1.616

1.620

1.616±0.04

Table 5: OD values of the respective protein concentrations at 490 nm (for Hexose sugar estimation)

Graph 4: Hexose (Glucose) standard graph

Calculations

The amount of wet cuticle taken is 0.352 gm for the estimation of the carbohydrates. From Hexose standard equation we have the regression equation as

Y = A + B x X

Where Y = OD value taken from spectrophotometer = 0.953

A = X- intercept from the graph = 0.006

B = slope of the line from graph = 0.041

X = (Y-0.006)/0.041

= (0.953-0.006)/0.041

= 23.675 µg of Hexose sugars/0.352 gm of cuticle

Result

The results forecast that 67.258 µg of Hexose sugars are present in 1gm of the cuticle isolated from the body of earthworm and approximately 0.067% of the Hexose sugars are present in the isolated cuticle collagen.

4.8.2 Total Lipid content

The final results predict that approximately 57 mg of lipid is extracted per 1 gm of cuticle isolated from the body of the earthworm, which shows that approximately 5.7% of lipids are present in the cuticle collagen.

5. DISCUSSION

The protein component of earthworm cuticle is associated with a very much larger amount of polysaccharide material than is found in the vertebrate collagens. The same is true of two morphologically distinct types of fibril from the mesogloea of the sponge Spongia graminea (Gross et al., 1956).

Earthworm cuticle has been shown to lack some of the main distinguishing features of a mammalian collagen fibril. Reed & Rudall (1948) report that the material does not show the familiar 640 Ao banding, visible in electron micrographs of collagen fibrils. Also it has been observed that the protein component of earthworm cuticle shows hydroxyproline, an amino acid which is found significantly in hydrolysates of vertebrate collagens. Earthworm cuticle also belongs to a growing number of collagenous proteins (Damodaran et al., 1956; Eastoe., 1957), which do not fit into the correlation between hydroxyproline content and hydrothermal-shrinkage temperature observed by Gustavson (1955) in a variety of fish collagens. It now seems unlikely that bonds involving specifically the hydroxyl group of hydroxyproline have a great stabilizing effect upon all collagen fibrils.

When isolated and purified cuticular fibrils of Lumbricus observed under Polarizing microscopy, the collagenous nature of the isolated fibrils is confirmed (Zuccarello., 1979). Study of negatively or positively stained isolated fibrils, under electron microscope resulted that they are cylindrical, unbranched and without periodic structure. In earthworms, the external and internal collagens seem to differ in structure (Watson., 1958., 1959). ACL (Anterior Cruciate Ligament) of rabbit shows fine fibrillar collagens under electron and light microscopy as narrated by Bayat et al., 2003, where these structures are unique and almost having morphological similarity with that of earthworm collagens. When soluble earthworm cuticle collagen molecules are subjected to the shearing forces of a flow birefringence instrument, they are broken into particles approximately half the original size (Maser., 1963).

Singleton (1957) has published some analyses of earthworm cuticles isolated from Allolobophora longa. In detail, these analyses are rather different from those reported above for Eisenia, whether this represents a species difference or is a result of the extensive pretreatment of cuticles. It is almost very clear that the Allolobophora cuticles appear to contain less polysaccharide than do the Lumbricus cuticles, which is also less than Eisenia cuticles.

6. SUMMARY & CONCLUSIONS

6.1 SUMMARY

The test organism was brought to the laboratory carefully and was grown under the laboratory conditions according to OECD guidelines.

The earthworms were taken in batches and were immersed in the dry ether for hydrolysis and then the cuticle was peeled off carefully with the help of forceps.

The total amount of cuticle peeled was quantified that about 4.97 gm cuticle was removed from 100 adult earthworms.

Isolation of the cuticle collagen was carried out and its characteristics such as total protein content, 4-Hydroxyproline content and SDS-PAGE gel electrophoresis were studied.

The structural studies of the earthworm cuticle collagen were studied using the LM and TEM.

The total macromolecules such as carbohydrates content (pentoses and hexoses sugars) and total lipids present in the isolated collagen were studied.

6.2 CONCLUSION

In agriculture, earthworm plays a very productive role in human life. As the products of Bovine, Porcine, Ovine, Avian and Piscean collagens are giving severe complications, biologists are looking for a different collagen source and in lieu to this, present study done for the search of safer collagen from earthworms. Easy extraction methods and immense availability of earthworm cuticle collagens can help in targeting certain important collagens for the benefit of mankind. The followers can utilize further biochemical, molecular and bio-physical tools for checking the homogeneity of earthworm collagens with the safer and compatible vertebrate collagens.

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