]. Cosmet. Sci., 58, 527-534 (September/October 2007) A simple improved method for protein extraction from human head hairs MI-OK HAN, JAE-AN CHUN, WOO-HYUP LEE, JIN-WOO LEE, and CHUNG-HAN CHUNG, Department of Biotechnology, Dong-A University, Busan 604-714, South Korea. Accepted for publication March 9, 2007. Synopsis This study was conducted to establish a simple efficient method for extracting the protein from human head hair materials, which can be a useful tool for the protein analysis applicable to various types of human head hairs. The method developed saves extraction time and effort considerably. The method includes four steps: cutting the hair samples into small pieces 1-2 mm in length, washing them with distilled water, incubating the hair samples in a buffer solution at 50 ° C for 24 hr, and finally filtering the incubated mixtures through three layers of nylon mesh. This method is reproducible and reliable. SDS-PAGE analysis of the hair protein extracted by this method shows a clear protein profile on the gel, which is frequently observed in other hair sources. Two smaller sizes of molecular weights are also detected with the SDS-PAGE analysis. Not commonly found in other hair sources, they seem to be other types of human hair proteins. INTRODUCTION Human hairs are comprised of approximately 80% protein, in which two large families, the intermediate filament protein family and the keratin-associated protein family, are formed (1-3). Proteins are key components in determining the shape of human hairs, and different types of proteins contain different amounts of disulfide bonds. Because hair proteins are very stable and resist enzymatic degradation, hair protein profiles can be utilized for characterization of various types of hairs derived from different sources, including hairs damaged by various hair treatments (4-6). Gerhard and Hermes (7) showed that there are eight characteristic polypeptide patterns in the human body and that head hair of an individual contains a specific polypeptide pattern. Moreover, the fact that the electrophoretic protein typing technique could be applied to hair genetic analysis and cosmetic sciences has been demonstrated by some workers (5-8). In addi­ tion, Shimomura et al. (9) showed that the polymorphic patterns of multiple keratin­ associated proteins in human hairs may be a useful tool with regard to biological identification between different individuals and different types of hair sources. The Address all correspondence to Chung-Han Chung. 527

528 JOURNAL OF COSMETIC SCIENCE findings mentioned above imply that human hair proteins could be used as an indicator in distinguishing the diverse status of human hairs as well as for basic human hair protein analysis. In comprehensive protein analyses of human head hairs, the initial concern is to obtain not only quality protein but also amounts sufficient to assay chemical and/or biochemical properties of the proteins in question by mapping/typing, purification, sequencing, hair structural and proteomic analyses, etc. To successfully apply these techniques to analysis of human hair proteins, quality proteins and large amounts of them are needed (10). Some problems, however, are frequently encountered in preparations of human hair protein. The major problems encountered are caused by the presence of highly cross­ linked disulfide linkages and detergents such as sodium dodecyl sulfate (SDS) and guanidine hydrochloride (11, 12). The presence of these substances in the extraction solution of hair protein has an inhibitory influence on the chemical and physical reac­ tions of the solution and thus affects hair protein analysis ( 11, 12). Another problem is that the isolation procedures are complicated and time-consuming. In general, the surface of human hairs is lipidized or contaminated with various chemical compounds, and thus some pretreatments have been performed with delipidizing or other reagents such as ethanol, petroleum ether, a mixture of chloroform/methanol, or a mixture of hexane/dichloromethane to eliminate the external lipid or the contaminated substances (7 ,8,10,11). These pretreatments lead to inefficient procedures in extracting human hair proteins. Thus, we have developed a simple extraction method for hair protein, accel­ erating the procedure by reducing the extraction steps. We report here on the detailed protocol. MATERIALS AND METHODS PREPARATIONS AND PRETREATMENTS OF HUMAN HEAD HAIR SAMPLES, BUFFER SOLUTIONS, AND TEMPORAL ANALYSIS Samples of human head hair shafts were collected from one woman volunteer to mini­ mize the experimental variations that may be caused by sampling sources such as age or gender. The collected hair shaft samples were cut into small pieces with a length of approximately 1-2 mm. These small pieces of the hair shafts were used for these experiments. In the first step of the experiment, three extraction buffers were employed to compare the effect of the buffers on the extraction of proteins from the hair shafts. Extraction buffer A was a solution of 25 mM Tris-HCl (pH 8.5), 2.6 M thiourea, 5 M urea, and 5% 2-mercaptoethanol. Extraction buffer B was comprised of a solution of 25 mM Tris-HCl (pH 9.5), 8 M urea, and 5% 2-mercaptoethanol. For extraction buffer C, a commercial extraction kit applied commonly in extracting proteins from various tissues was used. In the second step, three experiments were carried out to scrutinize the effects of the washing pretreatments of the sample hairs on the protein extraction. The first experi­ mental group of hair samples was prepared with 3 x washings, using distilled water as a control. The second group was treated with 75% ethanol for 30 s after 3x washings with distilled water and the drying of the hair samples. The small pieces of the hair