jo Soc. Cosmet. Chem., 46, 11-28 (January/February 1995) An investigation into the action of transglutaminase on human hair JOHN M. GARDNER, PAUL E. SWANSON, and B. VIRGINIA TORRES-LOPEZ, Central Research and Development, The Dow Chemical Company, Midland, MI 48674. Received August 25, 1994. Synopsis Enzymes may offer an attractive alternative to traditional chemical approaches in permanent modification or conditioning of hair. The goal of this research was to determine if glutamine residues on the surface of human hair were recognized as a substrate for guinea pig liver transglutaminase. Optical and isotope assays were developed and used to monitor specific activity. Traditional amine donor substrates were used in conjunction with control treatments and rinsing procedures to seek evidence of covalent modification. No conclusive evidence was found for biocatalytic activity of transglutaminase with virgin hair. An estimate based on literature data from a nonsoluble glutamine substrate indicated that the detection limit of the isotope assay was approximately two orders of magnitude more sensitive than required to verify reaction. The results appear to contradict previous work in which it was thought that transglutaminase cross-links endogenous glutamine and lysine residues on the hair surface. Reaction with hair in the present work may have been limited by the presence of the proposed fatty acid layer (F-layer) on the hair surface. Future work with transglutaminase might be directed toward applications that do not require hair to donate endogenous residues to the reaction. INTRODUCTION The two primary morphological regions of human hair are the cuticle and the cortex. The cuticle is an external, layered network of flattened cells that protects the cortex from damage (1,2). Shampooing, wet combing, dry combing, toweling, styling, and weath- ering are all known to contribute to cuticle degradation (3-5). Wet combing is believed to be the most damaging hair care operation performed on a daily basis (6,7). Cell fragments are exfoliated during combing, gradually exposing cortex and creating an area subject to fibrillation or fracture. Functional polymers can be formulated into products to reduce wet combing damage by improving lubricity (8-10). These materials are deposited during shampooing or conditioning and have been designed to be substantive but not accumulate from repeated use. Some attempts have been made to "permanently condition" hair using reactive materials such as certain protein hydrolyzates and their derivatives (11,12). These are not in wide use, however, because the cystine in hair 11

12 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS needs to be reduced for reaction to occur. This has limited practical applications to permanent wave formulations. Enzymes may offer an attractive alternative to traditional chemical approaches in per- manent modification or conditioning of hair. Enzymes catalyze chemical reactions in aqueous solutions, at ambient temperatures and in a neutral pH range. Protein disul- fide-isomerase (EC 5.3.4.1), for example, is known to catalyze the rearrangement of disulfide bonds in proteins (13). A hair treatment using this enzyme has been described but not commercialized (14). Hair protein contains numerous other side chain func- tionalities that are amenable to covalent modification (15). Therefore, our approach was to identify enzyme systems that could target other "modifiable" functionalities in hair, such as amino groups (lysine, arginine, or histidine), carboxylates (glutamic and aspartic acids), alcohols (serine, threonine, tyrosine), or amides (glutamine, asparagine). Due to the large size of typical enzymes and constraints for materials diffusing into hair (16), treatment activity would be expected to be confined to the hair surface. Transglutaminases (EC 2.3.2.13) were chosen for initial investigation of applications for covalent modification of human hair. This class of enzymes catalyzes a unique transami- dation reaction involving the e-amino group of lysine and the •-amide group of glu- tamine: O Protein Backbone O '•L3•s +NH3 (1) Two polypeptides are cross-linked through an amide linkage with concomitant release of ammonia. The amide linkage formed does not involve the e•-amino group of either peptide, and so it so commonly referred to as an isopeptide linkage. These enzymes increase the molecular weight of protein substrates and have been shown to play unique physiological roles in a variety of tissues from which they have been isolated (17-25). Human plasma transglutaminase (Factor XIII) and guinea pig liver transglutaminase are the two most studied enzymes in this class. The plasma enzyme is involved in the final stages of the complex blood clotting cascade (17-19), whereas the role of the liver enzyme is still not well understood (25). The guinea pig liver enzyme is, however, the only commercially available enzyme in this series (Sigma). Inasmuch as the properties of this enzyme have been well studied (26-28), and as glutamine was known to be a major component of hair cuticle (15), transglutaminase was a good candidate for experiments with hair. Several patent applications were recently disclosed claiming that damaged hair surfaces were "reconditioned" through application of formulations containing either guinea pig liver transglutaminase or a newly isolated, bacterially expressed transglutaminase (29- 32). Reaction was stated to occur through endogenous glutamine and lysine residues on the hair surface (31). Subjective consumer evaluations and limited laboratory data showed improvements in several hair properties, attributed to the action of the enzyme cross-linking the hair surface and improving its "microsurface" condition. No mecha- nistic evidence was produced, however, to support this theory. If hair is in fact a