International Journal of Biological Macromolecules 93 (2016) 172–178 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Transient structures of keratins from hoof and horn influence their self association and supramolecular assemblies Prachi Kakkar, Madhan Balaraman ∗ , Ganesh Shanmugam Central Leather Research Institute-Council for Scientific and Industrial Research, Adyar, Chennai-600020, India a r t i c l e i n f o Article history: Received 25 October 2015 Received in revised form 26 August 2016 Accepted 27 August 2016 Available online 28 August 2016 Keywords: Aggregation Keratin Secondary structure Particle size Supramolecular assembly a b s t r a c t Keratins as fibrous proteins, offer structural integrity to various tissues in providing the functional role of protection or load bearing. This work is a prelude to understand the structure − property correlation for a wide variety of keratins. The kinetics of aggregation of bovine hoof keratin (KF) and horn keratin (KR) were monitored by different biophysical methods. pH dependent studies indicated that initially both keratins existed in pre-aggregated form and the efficiency of aggregation decreased with increasing pH. The size of the aggregates was found to be larger in KF compared to KR. UV–vis and particle size analysis clearly revealed that the pre-aggregated forms of KF and KR dissociated to intermediate transient structures with smaller aggregate size, which acted as stronger nucleating agents for further self association of the keratins to form higher order supramolecular assemblies. Conformational analysis indicated that there was no significant conformational change during the aggregation of KF and KR. Morphology of the KF aggregates showed fractal arrangement while KR aggregates formed an ordered structure with no particular arrangement. To the best of our knowledge, this is the first report which shows an interesting and unique observation on changes in the structure during self-association of keratins. © 2016 Published by Elsevier B.V. 1. Introduction Studying the structure of a biological material can provide insights to understand its function and mechanical strength [1]. Biological systems strategically construct their structures by means of self-organization/self-assembly [2]. It has been demonstrated that by self-assembling, these biological systems can evolve towards higher order structures [3,4] in response to changing mechanical and structural requirements [5]. Folding of proteins to a unique structure is a remarkable phe- nomenon which enables them to generate enormous selectivity and diversity in their functions [6,7]. One such example is keratin, which is a structural protein. Keratins have evolved from lower eukaryotes to mammalian epidermal intermediate filaments [8]. Two major categories of keratins are − -keratins and -keratins whose presence and quantity is based on the structural require- ment of that species. It is interesting to note that in mammals, -keratins form the structural basis of epidermis and epidermal appendages (like, wool, claws, nails, horns and hooves), whereas, in case of birds, -keratins make up the stratum corneum of ∗ Corresponding author. E-mail addresses: bmadhan76@yahoo.co.in, madhan@clri.res.in (M. Balaraman). epidermis and reticulate scales [9–12]. Often keratins are referred to as dead tissues which are devoid of vascularization because ker- atinocytes die soon after producing keratin. Keratins are found in a variety of morphologies based on their functions which ranges from a water-resistant material (present in turtle shells) to a highly ordered, structurally robust and impact-resistant material (in case of hooves) [13]. Bovine hoof structure comprises of tubules embedded in inter- tubular material, oriented in longitudinal direction where keratin is formed in the circular lamellae surrounding the tubules [14,15]. On the other hand, horn keratin has a laminar structure consisting of flattened and curved epithelial cells filled with keratin [16]. Hooves and horns comprise of complex packing of tubules and intertubular material. This packing/arrangement varies in hooves and horns in such a way that the former gets appreciable mechanical strength to support large compressive and impact loads, while the latter becomes stiff and rigid for the defence of the animal [4,16,17]. Due to their varied functions, hoof and horn keratins are expected to portray different properties. Folding and aggregation are associated with proteins resulting in supramolecular assembly to perform their required functional and/or structural roles [18]. It is hypothesized that the starting reactant in protein aggregation is the monomeric form of the pro- tein which undergoes a required conformational change thereby http://dx.doi.org/10.1016/j.ijbiomac.2016.08.077 0141-8130/© 2016 Published by Elsevier B.V.