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M.Nirenjana*, T.N. Archana*, Anubhav Malhotra*, Swarna V Kanth#, B Madhan#, J. Raghava

Rao#, Balachandran Unni Nair#, S. Sadulla#, T Ramasami#
* - Department of Leather Technology, Allagappa College of Technology, Anna University, Chennai - 25
Central Leather Research institute, Adyar, Chennai-20
Key words: Alginic acid, crosslinking, collagen

Polysaccharides are abundant in nature and are profuse, non-toxic, biodegradable natural polymers
possessing high degree of functionalization. However to fulfil the demand for tailored application profiles,
native biopolymers are often modified. Alginic acid is one such polysaccharide that has been modified and
widely used in food ,phamaceutical, and medical industries. Alginic acid has been modified to dialdehyde
alginic acid and used as a tanning agent for the stabilization of collagen. Shrinkage temperature
measurements, collagenase activity, scanning electron microscopy, infrared spectroscopy and differential
scanning colorimetry techniques have been investigated.

1.Introduction most commercial tanning practices, as they result

in leathers for different end uses with good
Leather is one of the unique material which physical and organoleptic properties. In recent
posses characteristics like breathability, times because of the ecological concerns the
viscoelasticity which arises from the naturally leather industry is looking for an alternative
woven fibrous skin matrix. These characteristics tanning system. It faces serious disadvantages
of leather is well suited in several applications. due to the constraint of its discharge norms of
In leather processing the natural material skin is 2ppm[12]. The currently practiced chrome
being stabilized against degradation. The unit tanning procedures results in only 60-65% of the
process involved in the stabilization of skin into chromium being offered to the leather and a
leather is called tanning. There are various substantial amount of chrome is discharged into
chemical agents used for tanning. Currently the the effluent [13]. As of now there is no safe
tanning industry is dominated by the use of method of disposal method available for the
chromium salts. chrome effluent or the chrome tanned leather
products, which is a major concern. Hence
Hides and skins predominantly contain collagen, researchers throughout the world are looking for
the most abundant protein in animals. There are alternative tanning systems, which would result
19 different types of collagen [1], of which type I in leathers with good hydrothermal stability,
collagen is the main component of skin, tendon, strength characteristics, organoleptic properties
bone and other tissues. This protein is present as and very importantly eco-friendly. A cleaner and
chains wound in triple helical form [2], which sustainable tanning agent/system that can result
are organized into fibrils of great strength and in leather with good functional properties is the
stability [3]. The structure of collagen is current need of the tanning industry.
stabilized by inter- and intra-chain hydrogen
bonds [4] and by water mediated hydrogen Biomaterials, biodegradable polymers are
bonds [5,6]. important biomaterials, which are abundant in
occurrence possessing a high degree of
In olden days collagen stabilization was brought functionalisation. Alginic acid is one such
about by plant polyphenols, through multiple material, which has already established its
hydrogen bonds [7,8]. Some of the oligomers of applications in food, pharmaceutical, and
chromium were known to interact with collagen medical industries (Kennedy, Griffith, Atkins,
[9,10], bringing about irreversible matrix 1984; Mc Neely and Pettitt, 1973). Molecular
changes, thus imparting higher stability [11]. The design of these materials plays an important role
networks formed between collagen and tanning in determining their suitability in such
agents are covalent bonds, complex bonds applications. Alginic acid is a linear, binary
Coulombic forces, H-bonds and hydrophobic copolymer of 1,4-linked -L-guluronic acid and
interactions depending on the tanning system. -D-mannuronic acid [14], usually isolated from
Chrome tanning is a primary tanning system in a brown algae but is also present in some species
of bacteria [15,16]. Along the polymer chain the cheapest raw material with large number of
two residues are arranged in an irregular aldehyde groups in a single molecule.
blockwise pattern [17-19]. There are three types
of blocks: homopolymeric sequence of This paper presents the studies on the feasibility
mannurronate (MM blocks) and guluronate of using dialdehyde alginic acid as a tanning
residues (GG blocks) and a region where the two agent for stabilization of collagen.
residues alternate (MG blocks). The relative
proportions of these block types are affected by 2. Materials and Methods
several factors [20-22] such as the botanical 2.1. Materials
source, plant maturity, collection site and Type IA collagenase was obtained from SIGMA
seasonal variations. ALDRICH, U.S. The Alginic acid and other
In solution, alginates behave like flexible coils chemicals used for the process were obtained
[24]. However on interaction with metal ions, from S.D fine chemicals.
they form ordered structure as evidenced by their
X-ray diffraction patterns [25]. Attempts have 2.2. Methods
been made to covalently crosslink sodium 2.2.1.Periodate oxidation of alginic acid
alginate with gelatin and athylenediamine using 20 g Alginic acid and calculated amount of
water-soluble carbodiimide. Recently it is sodim metaperiodate were dispersed in 100ml of
reported that although higher molecular weight distilled water and stirred in the dark
alginate is non-biodegradable, its dialdehyde magnetically at 25C for 6 hrs to obtain
derivative is biodegradable [26]. Alginic acid is dialdehyde Alginic acid of different degree of
the only polysaccharide, which naturally oxidation. The degree of oxidation was followed
contains carboxyl groups in each constituent by determining the concentration of periodate
residue, and possesses various abilities for left unconsumed by iodometry after 6 hrs [31],
functional materials. To fulfill the demand for and the degree of oxidation was found to be
tailored end use applications the native 98%.
biopolymers are often need to be modified.
Tanning with aldehydes is known to mankind for 2.2.2 Fourier transform infrared (FT-IR)
a very long time and aldehydes such as spectra
formaldehyde, glyoxal and glutaraldehyde have FT-IR spectra of Alginic acid and dialdehyde
been used for stabilization of skin[27-30]. In the Alginic acid were acquired on a Impact 400 FT-
present study alginic acid is converted to IR instrument using a potassium bromide (KBr)
dialdehyde alginic acid and the crosslinking disc containing 1% finely ground sample.
ability of this modified biopolymer is studied.
2.2.3 Differential scanning calorimetry(DSC)
Thermal properties of Alginic acid and
dialdehyde Alginic acid were analyzed using
Dupont 2910 Differential Scanning Colorimeter.
All measurements in the instrument were
conducted under a nitrogen atmosphere. The
samples were scanned at 5C/min . The
temperature at the midpoint of the change in
slope of the DSC change was taken as the
Structural formula of alginic acid [23] structural change in Alginic acid to dialdehyde
Alginic acid with respect to thermal heat.
Dialdehyde alginic acid has been proved to be
biodegradable and toxologically accepted 2.2.4 Pretreatment of collagen
chemical [26]. Hence dialdehyde alginic acid has Collagen matrix of goat skin has been used for
the dual advantage of being a tanning material crosslinking experiments. The raw skin has been
and the leather products after usage will not have converted to acidified collagen (pickled pelt) at
disposal problems, as they can be made pH 3.0 after adopting a sequence of
biodegradable under certain conditions. In this operations/processes that is conventionally
context, the dialdehyde starch has immense carried out in the pretanning operation during
potential to be used as a tanning agent in leather leather manufacture [32].
processing as it is abundant and one of the
2.2.5 Crosslinking of collagen with dialdehyde form of hydroxyproline from insoluble collagen
alginic acid [38]. Aliquots of 750 l of supernatant of
Ten gram portions of acidified collagen matrix samples collected at different time interval were
have been brought to a pH of 8 by treating the withdrawn after centrifuging at 10,000 rpm for
collagen with 8% sodium chloride and 80% 10 min. The collagenase hydrolysate was
water in a bottle shaker at 8 rpm for 30 minutes hydrolyzed in sealed hydrolysis tubes with 6 N
at room temperature, 2% sodium formate as 10% HCl for 16 hrs. The hydrolysates were
solution has been added and the shaker has been evaporated to dryness in a porcelain dish over a
run for 30 minutes and then 3% sodium water bath to remove excess acid. The residue
bicarbonate as 10% solution was added in 3 free of acid was made up to a known volume and
feeds of 10 minutes in the running shaker. the percentage (%) of hydroxyproline was
Dialdehyde alginic acid at concentration of 10% determined using the method of Woessner [39].
has been used for crosslinking treatment to the This method of determining hydroxyproline
marked bottle shaker containing collagen matrix involves the oxidation of hydroxyproline to
at pH 8 and was run for 24 hours at 300C. At the pyrrole-2-carboxylic acid, which complexes with
end of the crosslinking period, the waste solution p-dimethylaminobenzaldehyde exhibiting
was collected and the uptake of dialdehyde maximum absorbance at 557 nm.
alginic acid was calculated. The crosslinked Hydroxyproline = (concentration in g/weight of
collagen samples were thoroughly washed and sample) x dilution factor. Soluble collagen =
aged for 24 hours. Hydrothermal stability and Hydroxyproline 7.4. % Collagen degradation =
Scanning Electron Microscopy experiments were (Weight of soluble collagen/Initial collagenous
carried out. The samples were further taken for weight) x 100. (Initial collagenous weight has
collagenase activity. been adjusted after taking into account the
amount of Dialdehyde alginic acid fixed)
2.2.6 Determination of hydrothermal stability
The temperature at which the collagenous fiber 2.2.8 Scanning Electron microscopic (SEM)
shrinks to one third of its original length is noted studies
as the shrinkage temperature of the fibre. The Dialdehyde alginic acid crosslinked sample
measurement of the shrinkage temperature were cut from the official sampling position [40]
determines the thermal stability of collagenous after the treatment process. The sample were
matrices. The shrinkage temperature is also fixed by soaking them with buffered formalin for
termed as hydrothermal stability as the 18 h. Samples were then dehydrated gradually
measurement is carried out in the presence of using acetone and methanol as per standard
water. The hydrothermal stability of native and procedure [41]. Samples were then cut into
dialdehyde alginic acid treated collagen was specimens with uniform thickness. The
measured using a Theis shrinkage meter by the specimens were then coated with gold using a
standard method [37]. JEOL JFC-1100E ion-sputtering device. A JEOL
JSM-5300 scanning electron microscope was
2.2.7 Collagenase Hydrolysis of the stabilized used for the analysis. The micrographs for the
collagen cross section were obtained by operating the
The enzymatic degradation of collagen adjusted SEM at an accelerating voltage of 20 kV with
to pH 7.0(native) and collagen stabilized with different lower and higher magnification levels.
dialdehyde alginic acid by collagenase was
analyzed by estimating the amount of 3. Results & Discussions
hydroxyproline released in the solution after
hydrolysis. The native and stabilized collagen There are two hydroxyls attached to carbons at
was treated with bacterial collagenase (Type IA) 2,3 position and one carboxyl in 6 position in
from Clostridium histolyticum. Collagenase each dehydrated glucose unit. There are many
treatment was carried out in 0.04 M CaCl2 reactive groups available in the alginic acid
solution buffered at pH 7.2 with 0.05 M tris HCl. molecule that can react with different chemical
The collagen: enzyme ratio was maintained at reagents. Periodate oxidation of alginic acid
40:1. The samples were incubated at a selectively cleaves the C2-C3 bond between the
temperature of 37oC. Samples were collected at two adjacent hydroxyl groups and the 1,2--diol
different time interval upto 96 hrs and stored in group in glucose is converted into a dialdehyde.
freezer. The cleavage of native and stabilized The advantage of periodic acid lies in the
collagen was monitored by the release of soluble specificity of its oxidation. It forms aldehydes
within the polysaccharide molecule but it does acid reduces, which again is an indication of the
not continue the oxidation of the polymers to low oxidation of alginic acid to alginic dialdehyde.
molecular weight water-soluble forms. The
extent of oxidation can be readily controlled, and
a complete range of aldehyde derivatives of a
starch is made available as the oxidation level
varies between 0 and 100%. At 100% oxidation
hydroxyl groups of each glucose residue in the
Alginic acid is converted to its corresponding
dialdehyde structure; at 50% oxidation half of
the glucose residues of the alginic acid are

3.1 Structural properties of alginic acid and

dialdehyde alginic acid
The FT-IR spectrum of alginic acidis shown in b
Fig. 1a. In the fingerprint region of the spectrum
of alginic acid three characteristic peaks appear
between 937 and 1111 cm-1; these peaks are
attributed to C-O bond stretching. The band at
948 cm-1 is assigned to 14 linkage. The peak
at 1038 cm-1 is characteristic of the
anhydroglucose ring O-C stretch. The peaks at
808 cm-1 and 787 cm-1 can be assigned to
mannuronic and guluronic acids. The band at
815 cm-1 is due to the polyguluronate structure
and the band at 880 presents the characteristic -
mannuronic residues. Another characteristic peak Fig. 1. FT-IT of a) Alginic acid and b) Alginic
occurs at 1630 cm-1, which could be a feature of acid dialdehyde
tightly bound water present in starch. The
spectrum also displays absorption peak at 1743 3.2 Thermal properties of alginic acid and
cm-1corresponding to the stretching band of the dialdehyde alginic acid
free carboxyl anions. An extremely broad band The thermal stability of the alginic acid and
due to the hydrogen bonded hydroxyl group (O- alginic acid dialdehyde were studied using DSC.
H) appears at 3403 cm-1 that is attributed to the The exothermal and the endothermal changes of
complex vibrational stretches associated with Alginic acid and oxidized alginic acid at the
free inter and intra molecular bound hydroxyl increase of constant temperature of 5oC were
groups which make up the gross structure of measured by DSC. Fig. 2a shows the results of
Alginic acid. The sharp band at 2926 cm-1 is native alginic acid. The peak at 124.18 C is due
characteristic of the C-H stretches associated to the thermal transition of Alginic acid. The
with the ring methane hydrogen atoms. DSC of the dialdehyde alginic acid shown in Fig.
2b indicates a peak at 135.53C, which is due to
Comparison of the spectrum of alginic acid with the thermal transition of introduction of
alginic acid dialdehyde (Fig. 1b) clearly indicates dialdehyde functionality. The DSC curve of
the deviations around 1600 1700 cm1, which dialdehyde alginic acid and Alginic acid indicate
could be due to the introduction of additional different thermal degradation characteristics
carbonyl stretching (C=O) of aldehydic between the native and the oxidized Alginic acid.
functional group. The occurrence of sharp band The dialdehyde alginic acid appears to be more
at 2926 cm-1 in the spectrum is associated with stable since the inception of degradation is at
the C-H stretching with the dialdehyde alginic higher temperature. This greater thermal
acid substituents. The strong band at 3404 cm-1 stability of the dialdehyde alginic acid molecule
(hydroxyl groups) of dialdehyde alginic acidis of may be due to the introduction of aldehyde
decreased intensity as the oxidation of alginic functionality by lowering the amount of
acid to dialdehyde alginic acid reduces the hydroxyl groups after oxidation of Alginic acid
number of hydroxyl groups as compare to alginic molecules. Thus the thermal stability is increased
as a large proportion of aldehyde functionality been established that a maximum offer of 10% of
prevails in the large Alginic acid molecule. aldehydes is sufficient for the stabilization of
a collagen. Hence the treatment of dialdehyde
alginic acid with collagen had been carried out at
10% offer, pH 8 for a period of 24 hrs. The
aldehydic functionality in the dialdehyde alginic
acid can covalently bind with amino groups of
the collagen and the hydroxyl groups of the
dialdehyde alginic acid can involve in hydrogen
bonding interaction with the functional groups
available in collagen matrix.

3.4 Hydrothermal stability of the stabilized

The shrinkage temperatures measured for the
acidified(pickled) skin and the dialdehyde alginic
b acid treated skin are given in Table 1. The
shrinkage temperature for the acidified collagen
fibres has been found to be 560C. Shrinkage
temperature is the temperature at which the skin
(collagen) matrix shrinks to one third of its
original length. The temperature at which the
shrinkage of collagen matrix occurs is highly
specific and occurs due to destabilization of the
protein. It is also termed as hydrothermal
stability. The hydrothermal stability of native
skin is around 600C, which is higher compared to
Fig. 2. DSC of a) Alginic acid and b) Alginic the acidified skin, because acidification alters the
acid dialdehyde IEP of the protein and the electrostatic
interaction due to salt bridges within the collagen
3.3 Interaction of collagen with dialdehyde matrix is comparatively stronger at IEP and
alginic acid hence the matrix can offer higher resistance to
The aldehydes are known to interact with the shrinking. The long-range ionic interactions and
amino groups of the protein [46]. The amino other non-covalent interactions are the cause for
groups are generally protonated at lower pH and ordering of the collagen molecules the fibre
hence aldehyde interaction with proteins is matrix. Hence the basic forces, which are
preferable at pHs above iso electric point (IEP) responsible for the high denaturation temperature
of the protein. The IEP of acid pretreated (alkali of this protein, could be attributed to such long-
followed by acid) collagen is 4.7 and above this range interactions. Any further increase in the
pH collagen will remain negatively charged and ordering of the collagen will also increase the
amino sites are available for the interaction of stability of the matrix against hydrothermal
aldehydes. Earlier studies indicate pH 8 is stress. Increase in resistance against
conducive for the interaction of aldehydes with hydrothermal stress is one of the important
collagen [43]. Also, periodate oxy starch, like aspects in the stabilization of collagen matrix. It
periodate oxycellulose, has been shown [44,45] is found that treatment of collagen with
to be extremely sensitive to degradation by dialdehyde alginic acid has resulted in increase
alkali, by virtue of the presence of dialdehyde of the hydrothermal stability of the collage
groups. Hydrolysis may take place either at the matrix considerably. As seen from the Table 1,
1,4-bonds or at the C2-O bonds, giving the hydrothermal stability of the collagen matrix
depolymerized and decomposition products, treated with dialdehyde alginic acid is found to
which may take part in the reaction in the be 800C, which is 240C more than is observed for
alkaline range [46]. Hence reaction pH 8 can acidified collagen. The interaction of dialdehyde
also favor such hydrolysis process which can alginic acid with collagen has further increased
result in the reduction of molecular weight and the long range ordering of collagen, which in
size and can favor the penetration of dialdehyde turn is responsible for the increase in the
alginic acid into the collagen matrix. It has also hydrothermal stability of the collagen matrix.
treated with alginic acid dialdehyde exhibited
Table 1: Hydrothermal stability of acidified and only 40% degradation. The interaction of
dialdehyde alginic acid treated goat skin dialdehyde alginic acid with collagen has
brought in changes in the active sites of collagen
Process Shrinkage because of which collagenase is not able to
temperature(C) express it activity of breaking down the
Acidified skin 56 collagenous matrix.
Dialdehyde alginic 80
acid treated skin Table2: % Collagen degradation (based on
release of hydroxy proline) from native and
alginic acid dialdehyde treated collagen after 72
3.5 Enzymatic Stability of dialdehyde alginic hrs of collagenase hydrolysis
acid treated Collagen:
Collagen is resistant to all enzymes except % Collagen
collagenase. Triple helical structure of the Degradation
collagen renders stability against degradation to
several proteinases other than collagenases [47]. Native Collagen 98
The stability of the dialdehyde alginic acid Collagen treated with 40
treated collagen matrix against enzymatic alginic acid dialdehyde
degradation has been studied by analyzing the
rate of hydrolysis of collagen on treatment with 3.6 Scanning Electron microscopic (SEM)
bacterial collagenase. Bacterial collagenase analysis of the native and stabilized collagen:
preferentially cleaves X-Gly (X is most Evaluation of opening up of fiber bundles by
frequently a neutral amino acid) bond of the - implicit approach was done using Scanning
Gly-Pro-X-Gly-Pro-X- sequence in the non polar Electron Microscope. Scanning electron
regions of the collagen molecule [48]. Bacterial micrographs of collagen matrix stabilized using
collagenases from Clostridium histolyticum the polyaldehyde showing the cross section at a
cleave collagen at multiple sites [49], whereas, magnification of x 600 is given in Fig. 3. The
mammalian collagenases from human-fibroblast SEM micrograph of dialdehyde alginic acid
cleave collagen only at a single site (leu-Ileu) treated collagen fibres as seen from Fig. 3
breaking it into a 3/4th and 1/4th fragment [50]. appears to be coated. The coating could be due to
Hence a treated collagen matrix exhibiting the presence of dialdehyde alginic acid around
stability against the activity of bacterial the collageanous fibres. Normally sample from
collagenase can exhibit high resistance to acidified collagen displays a very fine opening
degradation by any class of enzymes. Collagen up of fiber bundles because in pretreatment of
matrix treated with dialdehyde alginic acid has collagen with alkali followed by acid treatment,
been subjected to hydrolysis by collagenase and fiber opening is developed as an osmotic-driven
the resulting hydrolysates have been analyzed for splitting of fiber bundles and the hydrostatic
hydroxyproline released into solution. The pressure built inside the matrix keeps the fibers
hydroxyproline content is a direct measure of the apart from each other.
amount of collagen solubilised. The amount of
hydroxyproline released in the supernatant for
the native and dialdehyde alginic acid treated
collagen matrix samples subjected to collagenase
treatment for a period of 72 hrs is given in Table
2. From the table it is observed that skin treated
with alginic acid dialdehyde offers resistance to
degradation by collagenase. The stabilization
effect of the collagen fibers treated with
dialdehyde alginic acid, where marked decrease
in the degradation is observed as against the
untreated collagenous matrix. Native collagen
matrix has undergone extensive hydrolysis with
the treatment of collagenase. The native collagen Fig. 3. Scanning electron micrograph of the cross
exhibited 98% hydrolysis of collagen in the section (600X) alginic acid dialdehyde treated
above time period whereas collagen matrix collagen fibres
13. Chandrasekaran, B., Raghava Rao, J., Nair,
4. Conclusion B.C.U. and Ramasami, T. 1999, Journal of
Scientific Industrial Research, 58, 1-10.
The presented work clearly indicates that the 14. F.G.Fischer ,H.Dorfel, Z.Phys. Chem., 301
biopolymer alginic acid in oxidized form as (1955) 186.
dialdehyde alginic acid can be used in the 15. P.A.J.Gorin, J.F.T.Spencer, Can.J.Chem.,
stabilization of another biopolymer collagen. The (1966) 993.
treatment of collagen matrix with dialdehyde 16. A.Linker,R.S.Jones , J.Biol.Chem, 241 (1966)
alginic acid has improved the thermal and 3845.
enzymatic stability of collagen. Substantial 17. A.Haug,B.Larsen,O.Smidsrod,ActaChem.Scand,
increase in hydrothermal stability is observed on 21(1967)691.
treatment with dialdehyde alginic acid . The18. B.Larsen,O,Smidsrod,T.Painter, A.Haug, Acta
dialdehyde alginic acid treatment renders Chem.Scand,24(1970)726.
collagenous matrix resistant to enzymatic 19. A.Haug,B.Larsen,O.Smidsrod,Carbo.Res.,32,197
hydrolysis. The stability against heat and 4,217.
enzymatic hydrolysis are the main aspects to be20. J.W.A.McLee,L.Kavalieris,D.J.Brasch,M.T.Bro
considered for the permanent stabilization of wn,L.D.Melton,J.Appl.Phycology,4(1992)357.
collagen which is achieved in the process called 21. P.Gacesa,Carbo.Polym.,8,1988,161.
tanning. The dialdehyde alginic acid, a modified 22. O.Smidsrod,Carbo.Res.,13,1970,359.
polymer of alginic acid appears to be an 23. Phillips, Wedlock and Williams: Gums and
effective alternative material that can be used in Stabilizers for the Food Industry 5 (1990) by
the process of stabilization of collagen. permission of Oxford University Press.
24. O.Smidsrod,A.Haug,Acta Chem.Scand., 22,
5.References 1968, 797.
25. C.Sterling,Biochem.Biophys.Acta,26,1957,186.
1. Procop.D.J, Kivirikko.K.I; Molecular Biology,26. Bouhadir.K.H.,Lee.K.Y.,Alsberg.E.,Damm.K.L,
Diseases and potentials for therapy. Annu. Rev. Anderson.K.W.,Moony.D.J,Degradation of
Biochem.;1995,64,403-34. partially oxidized alginate and its potential
2. Ramachandran, G.N., Kartha G. 1955, Nature, application for tissue
174, 269. engineering.Biotechnol.Prog.,2001,17: 945-50.
3. Nimmi, H. E., Boca Raton, F. L. 1998, Collagen,27. Gustavson, K. H.; The Chemistry of tanning
CRC, 1. Process, Academic Presas, N.Y. p 244, 1956
4. Rich, A., Crick F. 1955, Nature, 176, 915. 28. Krysztof Bienkiewicz,; Physical Chemistry of
5. Ramachandran, G. N., Chandrasekaran, 1968, R. Leather Making, Robert E. Hrieger Publishing
Biopolymers, 6, 1648. Company, INC. Malabar, Florida, p. 374, 1983
6. Ramachandran, G. N., Sasisekaran, V. 1965,29. E. M. Filachione, M. L. Fein, E. H. Harris, F.
Biochem biophys Acta., 70, 11. P. Luvisi, A. H. Korn, W. Windus, J. Naghski.;
7. Tu, S.T., Lollar, R. M. 1950, Journal of Journal of American Leather Chemists
American Leather Chemists Association, 45, Association, 54, 668, 1959.
324. 30. George M. Sleichter, Robert M. Lollar;
8. Shuttleworth, S.G. 1952, Journal of American Journal of American Leather Chemists
Leather Chemists Association, 47, 603. Association, 49, 414, 1954.
9. Gayatri, R., Rajaram, A., Rajaram, R., 31. Guthrie RD. Periodate Oxidation. In: Whistler
Govindaraju, K., Rao, J. R., Nair, B.U., RL, WolframML, editors. Methods in
Ramasami, T. 1997, Proceedings of Indian carbohydrate chemistry. New York:Academic
Academy of Sciences (Chemical Sciences) 109, Press; 1953. p. 43247.
145. 32. Thorstensen, T. C. Practical Leather
10. Rao, J.R., Gayatri, R., Rajaram, R., Nair, B.U., Technology, 4th ed., 1993, Krieger Publishing
Ramasami, T. 1999, Biophys Biochem Acta, Co.: Malabar, FL.
1472, 595. 33. C.S. Wise and C. L. Mehltretter, 1958,
11. Santappa, M., Ramasami. T., Kedlaya, K. J. Analytical Chemistry, 30, 174-175.
1982, Journal of Scientific Industrial Research, 34. Nayudamma, Y., Thomas Joseph, K., Bose, S.
41, 616. M., 1961, Journal of American Leather Chemists
12. Standards for discharge of industrial effluents: Association, 56, 548.
Indian standards industrial effluents: Bureau of 35. Lenore S. Clesceri, Arnold E. Greenberg and
Indian Standards, 1985, IS-2490. Rhod IUC, Determination of Volatile matter,
Journal of Society of Leather Technologists and 45. Filachione, E. M., Clarke, I. D., Harris, E. H.,
Chemists, 7, 277, 2002. Fee, J., Witnauer, L. P., Naghski, J., and Boyd, J.
36. Es Trussell, Standards methods for the N.; Journal of American Leather Chemists
examination of water and waste water, 17th ed., Association, 52, 200, 1957.
American Public Health Association, 46. Krysztof Bienkiewicz,; Physical Chemistry of
Washington D. C, part 4500, 4-147 to 4-148, Leather Making, Robert, 1983, E. Hrieger
1989. Publishing Company, INC. Malabar, Florida, p.
37. Brodsky, R., Nutting G. C.; Journal of American 374 -378,
Leather Chemists Association, 44, 831, 1949. 47. Seifter S. and Harper E. (1971), The
38. Ryan J.N., and Woessner J. F., 1971, Biochem. collagenases, In: The enzymes, Boyer P.D., ed.,
Biophys. Res. Comm., 44, 144-149. Vol. II, Academic Press, New York, pp.649-697.
39. Woessner J.F., 1961, Arch. Biochem. Biophys., 48. Galardy R.E. and Grobelny D. (1983),
93, 440-447. Inhibition of collagenase from Clostridium
40. Echlin, P. In: Scanning Electron Microscopy; histolyticum by phosphoric and phosphonic
Heywood, V. H., Ed.; Academic Press: London, amides, Biochemistry, Vol. 22, pp.4556-4561.
1971; Vol. 4, p 307. 49. French M.F., Bhown A. and Van Wart, H.E.
41. Official methods of analysis; Society of Leather (1992), Identification of Clostridium
Technology and Chemistry: Herts, U.K., 1965. histolyticum collagenase hyperreactive sites in
42. Thiebaud,S.,Aburt,J.,Alric.,Borredon,E., type I, type II and type III collagens: Lack of
Bikiaris, D., Prinos,J., & Pnayiotou, C. J. 1977, correlation with local triple helical stability, J.
Journal of Applied polymer Prot. Chem., Vol. 11, pp.83-97.
Science,65,705 721. 50. Gomez D.E., Alonso D.F., Yoshiji H. and
43. Launer, H. F., Tomimatsu, Y. 1961, Journal of Thorgeirsson U.P. (1997), Tissue inhibitors of
organic Chemistry, 26, 541. metalloproteinases: Structure, regulation and
44. Meara, D., Richards, G. N. 1958, Journal of biological functions Eur. J. Cell Biol., Vol. 74,
American Chemists Society, 1204, 4508. pp.111-122.