ABSTRACT Organo-zirconium (Organozir) is a zirconium tan- ning salt complexed with organic moieties. The development of this tanning salt as a potential sub- stitute for chromium has been reported in our ear- lier work. In the present investigation, studies to understand the mechanism of tanning using organozir at the molecular level have been attempted. The thermal and enzymatic stability of collagen brought about by organozir have been studied using hydrothermal shrinkage measure- ment, differential scanning calorimetry (DSC) and hydroxyproline estimation respectively. It has been observed that collagen tanned with organozir exhibits a hydrothermal shrinkage temperature of 94 o C. The denaturation peak temperature observed from DSC, which indicates the helix ® coil transi- tion of collagen, also occurs at 94 o C. Mechanistic insights into the stability of collagen against colla- genase have been addressed by studying the con- formational changes occurring in collagen as a result of interaction with organozir using circular dichroism (CD). The interaction with organo-zir- conium complex results only in minor changes in the triple helical conformation of collagen. This indicates that organozir stabilizes collagen against collagenase degradation by blocking the active site in collagen. INTRODUCTION The present day research in leather sector is an eco-driven research. Many chemicals hitherto considered as harmless are soon becoming classified under the category of chemi- cals harmful to the environment. Chromium is one such chemical. Chromium(VI) is a known carcinogen and chromium(III) is considered as non-toxic. However, reports on chromium(III) bringing about DNA and protein damage under certain ligand environments are emerging. 1,2 Hence, alternatives to chromium for tanning are being sought actively. Our previous attempt to seek a potential alternative to chromium resulted in the development of an organo-zir- conium complex, organozir. 3,4 Mechanistic insight into tan- ning with organozir was attempted through indirect studies. 5 Tanning studies using deaminated and acetylated hide pow- der and urea treatment indicated possible involvement of both electrovalent and hydrogen bonding forces in the sta- bilization process of collagen. But the molecular level inter- action of organozir with collagen is yet to be elucidated. Hence, in this present investigation, an attempt has been made to unravel the molecular level interaction of organozir with collagen. Rat tail tendon (RTT), which predominantly contains Type 1 collagen, as in skin/hide, has been tanned with organozir to study the influence of the same on the thermal and enzymatic stability of collagen. The molecular stability of collagen arises from the interplay of a wide range of forces and the role of these forces in the stabilization process of collagen has been elucidated. 6-9 Native collagen is susceptible to attack by collagenase at physiological pH and temperature. 10-12 Differential scanning calorimetry (DSC) is a useful technique to study the thermal denaturation of collagen because the position, height, width, area and symmetry of the thermogram peak provide valu- able information about the denaturation process. Using DSC collagen denaturation can be studied in collagen fibres at different levels of hydration. 13-15 The conformational behav- ior of collagen in solution can be examined using circular dichroism (CD). The alterations brought about by organo- zirconium complex in the conformation of collagen and col- lagenase can thus be elucidated. The stability of the protein against enzymatic activity can be known by estimating the hydroxyproline content after subjecting the protein to enzyme degradation. Hence, in this study, the effect of cross-linking brought about by organozir on the thermal and enzymatic stability of collagen has been studied using dif- ferential scanning calorimetry (DSC), circular dichroism (CD) and hydroxy proline estimation. MATERIALS AND METHODS Sample Preparation Tails were excised from 6-month old male albino rats (Wistar strain) and frozen at -20 o C, the maximum storage time being 1 month. On removal from the freezer, tails were JALCA, VOL. 98, 2003 445 MOLECULAR LEVEL UNDERSTANDING OF T ANNING USING AN ORGANO-ZIRCONIUM COMPLEX by B. MADHAN, N. NISHAD F ATHIMA, J. RAGHAVA RAO* AND B. U. NAIR Chemical Lab, Central Leather Research Institute ADYAR, CHENNAI 600 020, INDIA *Corresponding author - email: clrichem@lycos.com