Improved binary high performance liquid chromatography for amino acid analysis of collagens S. Deb Choudhury, G. E. Norris Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand Abstract Resolution of complex amino acid mixtures obtained through hydrolysis of collagen (Type I) was achieved using pre column derivatisation and an octadecyl HPLC column. The use of a simple binary gradient in contrast to the usual complex gradient systems requiring precise control over mobile phase compositions ensured reproducible results. The selectivity offered by the stationary phase for the amino acids under the conditions used in this study makes the procedure less stringent for obtaining good resolution between amino acids differing slightly in their hydrophobicity. Keywords: Amino Acid analysis, HPLC, Collagen 1 Introduction Collagen is the most abundant protein in animal tissues and exists in various forms from skin and bone to basement membrane of capillaries. Collagens belong to a family of fibrous proteins, which provide the major biomechanical scaffold for anchorage of macromolecules and cell attachment. The distinctive nature of collagen poses a challenge for characterizing this protein using amino acid analysis. Collagen is characterized by a unique triple helical structure comprising of three polypeptide chains (alpha chains). In order for these chains to be wound around one another to form a super helix, the smallest amino acid, glycine (Gly) (having only a hydrogen atom as a side chain) must be regularly spaced at every third residue along each chain resulting in the repeating amino acid sequence, Gly-Xaa-Yaa. Xaa and the Yaa can be any amino acid, although they are often the imino acids proline (Pro), which is present in higher than normal concentrations in collagen, and hydroxyproline (Hyp). Another modified amino acid, hydroxylysine (Hyl), is also present in collagen, where it functions to stabilize side-by-side packing of collagen molecules through covalent aldol cross-links [1,2]. Collagen differs from other fibrous proteins (elastin, keratin and reticulin) by its high content of Hyp, Pro and Hyl, by its low tyrosine (Tyr) and sulphur content and by the absence of tryptophan (Trp). It also has a significantly higher content of polar amino acids than other fibrous proteins [1]. The amino acid composition of collagen varies between species, which is manifested in differences in the shrinkage temperature (the temperature at which sudden shrinkage in collagen fibrils occur as it is gradually heated in an aqueous medium) of collagen between species. This is primarily due to the number of Hyp residues present which in turn determines the number of water mediated hydrogen bridges formed, influencing the stability of collagen molecules. Because Gly is present in every third position in the amino acid sequence, it gives an enormous response during amino acid analysis. Added to the unusual distribution of a number of amino acids present in collagen, such as Pro, Hyp and Hyl, a careful selection of both stationary and mobile phases is required to provide a good selectivity and to prevent overlapping of closely eluting peaks. Separation and quantitation of acid hydrolysates of proteins and peptides is a common technique used by biochemists. Various methods are in use for quantitation of amino acids. Post column derivatisation of free amino acids with ninhydrin, after separation by ion-exchange columns was introduced by Moore and Stein nearly 50 years ago [3]. Since then, improvements in technology have resulted in reduced analysis times, increase in sensitivity of analysis and the ability to analyze limited sample amounts concomitant with increased use of reversed-phase chromatography and pre column derivatisation. Various pre-column derivatising agents such as ortho-phthalaldehyde (OPA) [4], phenylisothiocyanate (PITC)[5], 9- fluorenylmethyl chloroformate (FMOC-Cl) [6], dansyl [7] and 4-dimethylazobenzenesulfonylchloride (Dabsyl-Cl)[8], have their own disadvantages. OPA fails to react with secondary amino acids and the derivatives formed are unstable. Multiple derivatives are formed with FMOC-Cl and Dabsyl-Cl along with significant interference from the reagent itself. More recently a simple derivatisation procedure with highly sensitive and stable 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (AQC) has made this analysis a more robust technique. Sensitive fluorescence detection of highly stable urea adducts formed from the reaction of amino acids with AQC and good derivatising efficiency in presence of common buffer salts and detergents, provides an advantage over other derivatising agents [9]. 1st International Conference on Sensing Technology November 21-23, 2005 Palmerston North, New Zealand 332