Research articles

By Dr. Abbas B El-ta'alu , Prof. Eugene E Persky, , Dr. Natalya I Bulankina , Dr. Yury G Kot , Mrs. Eketerina V Kot , Mr. Alexander N Ponomarenko , Mrs. Tatiana V Kostina
Corresponding Author Dr. Abbas B El-ta'alu
V. N.Karazin Kharkiv National University, Dept. of Human Physiology, - Ukraine 61022
Submitting Author Dr. Abbas B El-ta'alu
Other Authors Prof. Eugene E Persky,
V. N. Karazin Kharkov National University, Dept. of Biochemistry , Kharkiv, Svobody Sqr. 4. - Ukraine 61022

Dr. Natalya I Bulankina
V. N. Karazin Kharkov National University, Dept. of Biochemistry, Kharkiv, Svobody Sqr. 4. - Ukraine 61022

Dr. Yury G Kot
V. N. Karazin Kharkov National University, Dept. of Biochemistry, Kharkiv, Svobody Sqr. 4. - Ukraine 61022

Mrs. Eketerina V Kot
V. N. Karazin Kharkov National University, Dept. of Biochemistry, Kharkiv, Svobody Sqr. 4. - Ukraine 61022

Mr. Alexander N Ponomarenko
V. N. Karazin Kharkov National University, Dept. of Biochemistry, Kharkiv, Svobody Sqr. 4. - Ukraine 61022

Mrs. Tatiana V Kostina
V. N. Karazin Kharkov National University, Dept. of Biochemistry, Kharkiv, Svobody Sqr. 4. - Ukraine 61022


Age, Collagen, Cross-linking, Gibb's free energy, Hydroxylation, Postnatal ontogenesis, Oxidative deamination, Thermal stability

El-ta'alu AB, Persky, EE, Bulankina NI, Kot YG, Kot EV, Ponomarenko AN, et al. The Role of Collagen Processing in Age-related Changes in the Thermo-stability of Connective Tissue Macromolecule - Collagen. WebmedCentral BIOCHEMISTRY 2011;2(11):WMC002392
doi: 10.9754/journal.wmc.2011.002392
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Submitted on: 29 Nov 2011 06:21:13 AM GMT
Published on: 29 Nov 2011 04:53:48 PM GMT


The relationship between the degree of hydroxylation and oxidative deamination of e-NH2-groups of lysine and hydroxylysine in collagen, and the thermal stability of this protein’s sub-molecular structures in the skin of Wistar rats in their postnatal ontogeny was in vitro studied. It has been shown that, rising towards the age of 3 months, the content of free e-NH2-groups in collagen continuously remains constant, while that of free aldehyde groups (COH-groups), starting from 1-month steadily decreases with age. Accordingly, intermolecular cross-linking in collagen’s sub-molecular structures is reduced during the period between 1 to 3 months, and afterwards continuously increases up to 24 months of age. The content of hydroxyproline in collagen is continuously reduced in postnatal ontogenesis. The combined effect of both effects leads to a decrease in the thermal stability of collagen sub-molecular structures in the skin during the period from 1 to 3 months, and subsequently rises up to 24 months old.


Sub-molecular collagen formations are one of the most important structural elements non-mineralised types of connective tissue. The level of structural stability of these structures largely determines connective tissue’s functional properties, primarily mechanical ones.  In most part of postnatal ontogenesis, structural stability of collagen formations increases, and this is regarded as one of the mechanisms of adaptation of mechanical properties of the connective tissue to increases muscle strength and mass of the organism during the process its growth and development. However, a short period in early postnatal ontogenesis exists, during which structural stability of these structures does not increase with age, but instead decreases, and continuously rises afterwards[10, 12; 15]. This phenomenon has not yet been explained.
Naturally, the reversal or inversion of the direction of age-related changes in the structural stability of sub-molecular structures in early postnatal ontogenesis must be due to certain specific changes in the structure of collagen molecules, which occur during this period.
One of the best methods by which integral structural stability of collagen formations can be evaluated is by investigating the level of their thermal stability. Thermal stability of collagen structures is determined by several factors: the steric properties of imino acids and the relative content of hydroxyproline in them, hydration of triple helices of molecules, the presence of hydrogen, as well as intra- and intermolecular covalent cross-links[2, 3, 4, 6, 8, 11]. Out of all these factors, distinct age-related changes in collagen structure are found only in the degree of their cross-linking, and in the content of hydroxyproline[1b; 13]. Both of these structural parameters are the result of post-translational modification of the primary structure of polypeptide chains of collagen by the processing enzymes prolylhydroxylase and lysyloxydase[10; 16].
While first enzyme hydroxylates pyrrolidine cycle of proline in the 4th position, under the action of the second, oxidative deamination of e-NH2-groups of lysine and hydroxylysine with the conversion of these residues into allyzyl and hydroxyallyzyl that contain aldehyde-group in place of e-NH2-groups. Subsequently, cross-covalent bonds are formed through the interaction of either two COH-groups (intra-molecular cross-linking - aldole bond -C = C) or e-NH2-groups with COH-groups (intermolecular cross-linking - aldimine bond -C = N-).
The aim of the present work was to elucidate the relationship between the degree of hydroxylation of proline, as well as oxidative deamination of e-NH2-groups of lysine and hydroxylysine and structural stability of sub-molecular structures of collagen in the skin of rats at different stages of their postnatal ontogenesis.

Materials and Methods

Investigations were carried out on skin collagen of 1 -, 3 -, 12 - and 24-month-old Wistar rats, which were kept in standard conditions in the vivarium of V. N Karazin Kharkiv National University, Ukraine. Skin samples weighing about 800 mg were taken from the dorsal part of the animals after their decapitation using Sodium thiopental anesthesia. In carrying out all experiments, a clearance was obtained from the authority concerned in the V. N Karazin Kharkiv National University, Ukraine, on the rules on treating animals in accordance with International principles of the European Convention «On protection of vertebrate animals used in experiments and other scientific works», and standards of biomedical ethics in accordance with the Law of Ukraine «On protection of animals from man-handling» were followed.
Thermal stability of collagen sub-molecular structures was evaluated thus, cleaned from subcutaneous fat layer and hair, skin samples were heated in distilled water at 60°C (20 mg of fresh tissue in 1 cm3 of H2O) in an incubator UT-Gibb’s free energy. Destruction of collagen structures was assessed by their solubility, by determining the amount of hydroxyproline released into solution after 1, 3, 5, 10, Gibb’s free energy, 20, 25, 30, 45, 60, 90 and 120 minutes of heating. Solubility was calculated as the content of hydroxyproline released into solution, to its initial content in samples, and expressed as %.
Using the obtained values, we plotted kinetic curves on the degree of solubility of collagen structures against time. Rate constants were determined from the tangent of slopes in the curves of corresponding areas of the kinetic curves with half their heights, and Gibb’s free energy of the process of the destruction calculated using the modified Eyring’s equation[9]: (Illustration 1).
The degree of proline hydroxylation was determined after skin samples were incubated in Ringer-Kreb’s medium in the presence of 3H-Pro (Amersham) with radioactivity of 0.4 MBk /cm3 for 6 hours at 37°C. Newly synthesised collagen in the incubated samples was extracted from incubation solution by 1M solution of NaCl. The degree of proline hydroxylation in collagen was judged by the relative radioactivities of ( Illustration 2), which was measured with Beckman’s counter LC 7800[7]. Error in determination was ± 5%.
The degree of oxidative deamination of lysine and hydroxylysine was evaluated by way of the contents of free e-amino and aldehyde groups, which were determined in collagen extracts by the methods[1a] and[5], respectively.
Analytical determination of hydroxyproline in samples was carried out by the method [14]. The curves shown in illustration 3 are typical of a series of repeated measurements (at least 8 - 9 samples in each series). Obtained values were statistically worked out using the program ‘Origin Pro 8.0’.

Results and Discussian

Illustration 3 shows kinetic curves of collagen release into solution, i.e. of collagen structures destroyed through heating in water of skin samples of animals of different ages, and table 1 - calculated from these curves, quantitative parameters of the destruction process. As can be seen, kinetic curves made up of two regions characterized by different slope at all ages.
In trying to explain this phenomenon, we note that, collagen molecules in sub-molecular formations are in different stages of polymerization and degradation. Consequently, these structures occupy an intermediate position between non-cross-linked and fully cross-linked collagen molecules that are composites of sub-molecular structures. In essence, they represent a «skeleton» made up of fully cross-linked molecules, coupled with recently synthesized molecules or with molecules currently under the stage of physiological degradation and weakly bound with it.
Apparently, the two portions on the kinetic curves (Illustration 3) correspond to processes of destruction of various areas in collagenous sub-molecular structures. The first portion is steeper, reflecting the denaturation and release into solution of denatured collagen molecules that are weakly bound to sub-molecular formations, including molecules that are recently synthesized and are in the stage of degradation. The second portion on the kinetic curves are flatter, reflecting the destruction and release of particles into incubation solution, molecules that more strongly bound, that were synthesized much earlier and included in the «skeleton» of sub-molecular structures.
In a qualitative similarity of the kinetic curves, they demonstrate striking age-related features of both processes.
In the first stage of destruction, the rate of collagen denaturation, which is proportional to the slope of kinetic curves, rising during the period from 1 to 3 months, subsequently continuously decreased up to  24-month of animals’ age. In the same manner and time course of changes in the first stage, as measured by the inflection point. At the same time, Gibb’s free energy of the destruction of collagen structures varies in the opposite direction – it decreases from 1 to months, then begins to rise and this increase continues until the age of 24-months.
In the second stage, neither the rate destruction nor the free Gibb’s energy of destruction of collagen in the skin depends on the age of the animals. This is due to the fact that the stiffness of fully formed cross-linked by covalent chemical bonds «skeleton» of collagen sub-molecular structures is closer in the animals, regardless of their age. However, the content of denatured collagen, which was released into solution in the second stage of destruction, depends on the age of the animals, increasing during the period from 1 to 3 months and then decreasing up to 24 months of age, as in the first stage of the hydrothermal treatment of the skin. This coincidence is explained thus, in equal rate of destruction, the rise of the release of denatured collagen into solution is constant and its total amount depends on the number of destroyed molecules released into solution at the end of the first stage.
Thus, according to the obtained data, the structural stability of the sub-molecular structures in the matrix of the skin of animals is decreased during the period from 1 to 3 months, after which it begins to rise (Illustration 4). The continuous rise in the process lasts up to 24 months of age. Age-related changes in the degree of hydroxylation and oxidative deamination of lysine and hydroxylysine are presented in (Illustration 5) and (Illustration 6).
As can be seen from the data in illustration 5, the degree of hydroxylation of proline in newly synthesized collagen decreases with age of the animals, indicating a decrease in the activity of  prolylhydroxylase throughout their postnatal ontogenesis.
The activity of lysylhydroxylase similarly decreases with age. Indeed, in 1-month old (Illustration 6), the content of aldehyde groups (COH-) of allysine and hydroxyallysine in newly synthesized collagen is more than by almost 5 times the content of e-NH2-groups. Subsequently, this difference decreases and at 24 months of age, the content of amine and aldehyde groups is virtually the same.
Free e-NH2- and COH-groups are potential precursors of cross-linking of covalent bonds. The probability of their mutual pair wise condensation depends on the concentration of the groups [6;.16]. Therefore, during the period from 1 to 3 months, cross-linking in collagenous sub-molecular structures must occur mainly due to the interaction of COH-groups with each other leading to the formation of aldoles – intra-molecular cross-links. After 3 months of age, due to a decrease in the activity of lysyloxylase and an increase in the relative content of e-NH2 – groups of lysine and hydroxylysine in collagen sub-molecular structures, due the interaction of these groups with COH- ones begin to form mostly intermolecular bonds of aldimine type. These age-related dynamics in collagen processing enzyme activities explains the inversion or reversal of changes in the thermal stability of collagenous sub-molecular structures in the skin during the period from 1 to 3 months of age. Thus, during the period from 1 to 3 months, the decrease in the integral thermal stability of collagenous sub-molecular formations in the skin is defined by the decrease in hydroxyproline content in the molecules, as well as the small formation of intermolecular cross-linking, linking them as an entity. After 3 months of age, the density of intermolecular cross-linking of collagen molecules in sub-molecular formations increases, which leads to an increase in their thermal stability as a whole. The decrease in the thermal stability of individual collagen molecules at the expense of subsequent decrease in them content of hydroxyproline is overlapped by the effect of intermolecular binding at sub-molecular level.


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