Electrophoresis 1991, 12, 451-453
Atsushi Nagai Hideou Komoriya Yasuo Bunai Sadao Yamada Xiuyan Jiang Isao Ohya Department of Legal Medicine, Gifu University School of Medicine, Gifu
IEF patterns of dyed and bleached halr proteins
45 1
Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach,are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach.
Hair dyes, which aim at altering the natural hair color, are generally classified into three types: temporary, semi-permanent, and permanent, according to their chemical action on the hairand the kind ofpigment used inmaking the dyes [l]. Further, permanent hair dyes are mainly divided into two types: metallic and oxidative dyes, with oxidative dyes still further divided into two subtypes: alkaline oxidative dyes and acidic oxidative dyes, according to their pH strength. Hair bleaches, which also affect the color of the hair, are used to decolorize the melanin of the hair and, like permanent hair dyes, they also possess a lasting effect [l]. Hair dyes, especially oxidative dyes, and hair bleaches have been found to induce morphological damage such as stripping the hair cuticle and exposing the hair cortex [2]. Hair dyes and hair bleaches, moreover, have been known to induce chemical denaturation of the hair proteins and to cause the formation of abnormal amino acids [2].However, only a few electrophoretic studies have been pursued with regard to the effect of hair dyes and bleaches on hair proteins [3,4]. To add to such electrophoretic investigations, in this paper we report on the effect of various hair dyes and bleach on the isoelectric focusing (IEF) patterns of the hair proteins. The dyes and bleach used for this study are listed in Table 1. Hair samples (hair shafts from the scalp) without prior hair cosmetic treatment were obtained from 31 healthy Japanese adults. The samples were washed with petroleum ether and ethanol, after which separate samples were treated with one of the listed dyes or the bleach in vitro, according to the instructions for each product. The subsequent extraction of the non-carboxymethylated fibrous proteins from both the treated hair samples and untreated hair samples was performed according to the method of Carracedo et al. [5]. Hair fragments (1 mg) were suspended in 100 pL of 0.05 M Tris-HC1, pH 9.3, containing 8 M urea and 0.05 M dithiothreitol (DTT), for 24 h at room temperature, with occasional shaking. The suspension was then centrifuged at 2500 rpm for 5 min and, to each 5 pLof resulting supernatant, 1 pL of 0.1 M DTT was added at least 10 min before IEF was initiated. Protein concentrations of the supernatant were determined by the method of Lowry et al. [ 6 ] , modified by Ross and Schatz [7], with bovine serum albumin as standard. Correspondence: Dr. Atsushi Nagai, Department of Legal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan Abbreviations: C, relative percentage of cross-linker in a polyacrylamide gel; IEF, isoelectric focusing; T,total monomer concentrations in polyacrylamide gel
0VCH Verlagsgesellschaft m h H , D-6940 Weinheim, 1991
IEF was performed in 0.5 mm thick polyacrylamide slab gels (5.0% T, 2.8% C) with 2.0% Ampholine carrier ampholytes (pH 4.0-6.5) and 4 M urea, using a Multiphorchamber (LKB,Bromma, Sweden) and a power supply, Model 3000/ 300 (Bio-Rad, Richmond, CA). Polymerization was carried out with ammonium persulfate and N,N,N', N' -tetramethylenediamine at room temperature. The electrode solutions were 1 M NaOH for the cathode and 1 M H,PO, for the anode. The gels were prefocused at 2 W constant power for 30 min at a cooling temperature of 4°C. Forty pL samples, without adjusting protein concentrations, were applied to Whatman 3 MM filter papers (10 X 10 mm) at a distance of 2 cm from the cathode and focusing was continued for 1 h. Then the papers were removed and focusing was continued at 4 W (maximum voltage of 1600 V) for an additional 180 min. After IEF, the gels were fixed in 20% trichloroacetic acid for 30 min and rinsed in a destaining solution (50% ethanol and 17% acetic acid) for 30 rnin with shaking. The gels were then stained with Coomassie Brilliant Blue R-250 for 1 h at room temperature and destained until sufficient contrast in the bands was achieved. The pZ values were determined by directly measuring the pH on the surface of the gel using a pH meter (Corning, NY, USA) with a microelectrode (Toko Chemical Industries, Tokyo, Japan). First, the IEF of the extracted proteins from 31 untreated hair specimens revealed that all the IEF patterns were exactly the same. Carracedo et al. [5] have reported electrophoretic variants of human hair proteins without carboxymethylation by IEF and by silver staining. Gerhard [8],Gerhard and Hermes [4], and Schimkat et al. [9] have also reported variants of human hair proteins without carboxymethylation on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Even so, we were unable to find any variants in our specimens on using the present method. In Fig. 1, the IEF results of the extracted proteins from dyed and bleached hair are shown. Note that the protein patterns of hair treated with a temporary dye (color spray) or a semi-permanent dye (acidic color) were identical to the protein patterns of untreated hair (Fig. 1, lanes 1-3). However, the IEF patterns of protein extracts from hair that was treated with a permanent hair dye (metallic, or alkaline oxidative, or acidic oxidative), or with a hair bleach were distinctly different from the pattern of untreated hair proteins. Additionally, the hair protein patterns of hair that was treated with an alkaline oxidative dye were identical to the pattern of the extracted proteins from bleached hair. In both types of patterns, a distinct band was observed at pH 5.2. Moreover, compared with the IEF patterns of the untreated hair proteins, the bands at pH 4.0-4.3 and pH 5.1 0173-0835/91/0606-0451 $3.50+.25/0
452
Electrophoresis 1991, 12, 451-453
A. Nagai et al.
Table 1. List of hair dye and hair bleach uscd for this study Effect on hair Temporary Semi-permanent Permanent
Bleaching
a) b) c) d) e)
Hair dyes and bleach used
Commercial name (Color)
Color spray Acidic color Metallic dye Alkaline oxidative dye Acidic oxidative dye Hair bleach
Ford Color Coat Spray (sepia) Color Story (madder-red) Marron (black) Simone Hair Color (safari brown) Real Care Tone (light brown) Sirnone Bleach
Source
4 b) c) d) el d)
Ford Hair Cosmetics, Osaka, Japan Arimino, Tokyo, Japan Yamahatsu Sangyo, Osaka, Japan Hoyu, Nagoya, Japan Real Chemical, Tokyo, Japan
were more intense, while the band at pH 5.3 was weaker (Fig. 1, lanes 5 and 7). As for hair that was treated with the acidic oxidative dye, the bands at pH 4.8-5.2 were weak (Fig. 1,lane 6).With respect to hairtreated with the metallic
1
2
3
4
5
6
7
Figure 1. IEF patterns of extracted proteins from hair treated with hair dyes or bleach. Lane (1) untreated; (2) color spray-treated; (3) acidic color-treated; (4) metallic dye-treated; (5) alkaline oxidative dye-treated; (6) acidic oxidative dye-treated; and (7) bleach-treated hair.
1
2
3
4
5
6
Figure2. IEFpatterns ofextracted proteins from hair treated with an alkaline agent or an oxidative agent. Lane (l),unreated; (2) 28% ammonium hydroxide-treated; ( 3 ) 6% hydrogen peroxide-treated; (4) with an equivalent mixture of 28% ammonium hydroxide and 6 % hydrogen peroxide; (5) alkaline oxidative dye-treated; and (6) bleach-treated hair.
dye, the band patterns were faint except for the pH 4.0 band andafewweakbandsat pH4.3-4.6(Fig. l,lane4).Changes in the IEF patterns were exactly the same for all the samples of the present study. Average protein concentrations of extracts from treated or untreated hair were as follows: untreated, 6.24 mg/mL; color spray-treated, 6.23 mg/mL; acidic color-treated, 6.41 mg/mL; metallic dye-treated, I .23 mg/mL; alkaline oxidative dye-treated, 9.13 mg/mL; acidic oxidative dye-treated, 6.80 mg/mL; bleach-treated, 8.00 mg/mL. Both alkaline oxidative dyes and hair bleaches contain an alkaline agent (mainly ammonia) and an oxidative agent (mainly hydrogen peroxide) [l].The former agent is used to make the hairs swell for easy permeation of the oxidative pigments and to decompose hydrogen peroxide into oxygen and hydrogen [I], whereas the latter agent is used to convert the aromatic amines in oxidative dyes into oxidized pigments and to decolor the melanin of the hair by its oxygen [l].It is further known that these agents chemically damage the hair proteins [2, 101. Therefore, to investigate whether these agents have an effect on the IEF hair protein patterns, the samples were either treated with 28 O/o ammonium hydroxide, 6% hydrogen peroxide, or an equivalent mixture of these solutions for up to 30 min, following the specific instructions on the use of each dye. The IEF hair protein patterns extracted from these treated hairs are shown in Fig. 2. The IEF hair protein patterns of hair treated with only ammonium hydroxide orwith only hydrogen peroxide were similar to the IEF patterns of the untreated hair proteins (Fig. 2, lanes 1-3). However, the IEF hair protein patterns of hair that was treated with an equivalent mixture of these two chemicales were similar to the IEF hair protein patterns of hair that was treated with an alkaline oxidative dye or a hair bleach (Fig. 2, lanes 4-6). These results indicate that the changes in the IEF hair protein patterns, observed after treatment with an alkaline oxidative dye or a hair bleach, were caused by a combined effect of an alkaline agent and an oxidative agent. The weak IEF patterns of hair protein treated with a metallic dye result from a distinctly lower protein concentration of the extracts. The concentration of about one-fifth of that from untreated hair is probably due to some components of the metallic dye, decreasing protein solubility. The cause for the change in the IEF pattern in hair treated with an acidic oxidative dye remains to be elucidated. Some reports state that hair cosmetic treatment, i.e., hair dying, bleaching and/or permanent waving, obstruct the detection of electrophoretic variants of the hair proteins [3,4].
Electrophoresis 1991, 12, 453-456
Quantification of proteins by colloidal silver
In the present study, we were able to demonstrate that metallic dying, oxidative dying, and bleaching induce changes in the IEF patterns and in the intensity of the hair protein bands. Furthermore, these results indicate that hair treated with a permanent hair dye and hair that is bleached can be differentiated from untreated hair by its characteristic IEF patterns and that metallic dyed hair as well as acidic oxidative dyed hair can be distinguished from hair that has been treated with other dyes. It was not clarified whether these effects accumulative with prolonged use of hair dyes or a hair bleach, are reversible following cessation, or complementary when these products are used in sequence. These results may prove useful for forensic cases requiring hair identification. This work was supported in part by a Scientific Research Grant (No. 01 770383)from the Ministry of Education, Science, and Culture, Japan. Received December 25, 1990
Pavel Draber Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague
453
References [ l ] Watanabe, Y., Tamura, T., Harada, H. and Yagihara, Y., Hairscience (in Japanese), Japanese SocietyofHairScience,Tokyo 1986,pp. 117154. [Z] Watanabe, Y., Tamura, T., Harada, H. and Yagihara, Y., Hair Science (in Japanese), Japanese Society of Hair Science,Tokyo 1986, pp. 95116. 131 Zahn, H. G., Bindewald, I. and Marshall, R. C.,J. For. Sci. SOC.1984, 24, 432. [4] Gerhard, M. and Hermes, M., Electrophoresis 1987. 8, 490-492. [S] Carracedo, A. Concheiro, L. and Requena, I., For. Sci. Int. 1985, 29, 83-89. [6] Lowry, 0. H., Rosebrough,N. J., Farr,A. L. and Randall, R. J.,J.B i d . Chem. 1951, 193, 265-275. [7] Ross, E. and Schatz, G., Anal. Biochem. 1973, 54, 304-306. 181 Gerhard, M., Electrophoresis 1987, 8, 153-157. [9] Schimkat, M., Baur, M. P. and Henke, J., Hum. Genet. 1990,85,311314. [lo] Toda,J.andOkamoto,K.,in:Arao,T.,Takashima,I.,Toda,J.,Hori,Y. and Okamoto, K. (Eds.), Medicine of Hair (in Japanese) Bunkodo, Tokyo 1987, pp. 280-296.
Quantification of proteins in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using colloidal silver A rapid and simple assay for quantification of proteins in sodium dodecyl sulfatesample buffer is described. Proteins are bound to a nitrocellulose membrane, stained with colloidal silver, and quantified by transmission densitometry. The staining requires 1 pL of protein sample and takes less than 20 min. Good linearity between the staining intensityand amount ofproteins is in the range of5-100 ng.
A quantitative analysis of proteins separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis often requires measurement of protein concentration in the SDS-sample buffer. When only a limited amount of material is available, a sensitive method is necessary for protein quantification. The use of nitrocellulose (NC) membranes for immobilization of macromolecules is well documented; bound polypeptides/proteins can be stained and quantified by densitometry [l-61. Silver staining [2] or AuroDye staining [3,5] of NC membrane makes it possible to detect protein quantities in the nanogram range. However, staining with AuroDye, although simple, requires several hours, and silver staining, based on a combination of silver photodevelopment and chemical development, is a multi~
~
Correspondence: Dr. Pavel Driber, Institute of Molecular Genetics, Czechoslovak Academy of Sciences,Videfiska 1083,CS-142 20 Prague 4, Czechoslovakia
Abbreviations: BSA, bovine serum albumin; NC, nitrocellulose; SDS, sodium dodecyl sulfate
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1991
step procedure. It has been shown that proteins electrophoretically transferred onto NC membranes could also be detected with high sensitivity by staining with colloidal silver [7]. This paper describes a simple and rapid quantification of proteins in SDS-sample buffer [8] by spotting the samples onto NC membranes and staining the dots with colloidal silver. NC membranes BA 85, BAS 85 (Schleicher and Schuell, Dassel, Germany), Synpor 6 (Synthesia; Prague, Czechoslovakia) or MSI NC membrane (MSI, Westboro, MA) with 0.45 V r n pores were used. All substances were of analytical grade and were obtained from Serva Feinbiochemica (Heidelberg, Germany) or Lachema (Brno, Czechoslovakia). U1trapure water from a Millipore water purification system was used throughout the assay. Bovine serum albumin (BSA, Cat. No. 11923) and lysozyme from chicken egg white (Cat. No. 28260) were supplied by Serva, bovine y-globulin (Cat. No. G SOOS), chicken egg albumin (Cat. No. A 5503), a-chymotrypsin from bovine pancreas (Cat. No. C 3142), aprotinin from bovine lung (Cat. No. A 1153), 0173-0835/91/0606-0453 $3.50+.25/0
Atsushi Nagai Hideou Komoriya Yasuo Bunai Sadao Yamada Xiuyan Jiang Isao Ohya Department of Legal Medicine, Gifu University School of Medicine, Gifu
IEF patterns of dyed and bleached halr proteins
45 1
Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach,are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach.
Hair dyes, which aim at altering the natural hair color, are generally classified into three types: temporary, semi-permanent, and permanent, according to their chemical action on the hairand the kind ofpigment used inmaking the dyes [l]. Further, permanent hair dyes are mainly divided into two types: metallic and oxidative dyes, with oxidative dyes still further divided into two subtypes: alkaline oxidative dyes and acidic oxidative dyes, according to their pH strength. Hair bleaches, which also affect the color of the hair, are used to decolorize the melanin of the hair and, like permanent hair dyes, they also possess a lasting effect [l]. Hair dyes, especially oxidative dyes, and hair bleaches have been found to induce morphological damage such as stripping the hair cuticle and exposing the hair cortex [2]. Hair dyes and hair bleaches, moreover, have been known to induce chemical denaturation of the hair proteins and to cause the formation of abnormal amino acids [2].However, only a few electrophoretic studies have been pursued with regard to the effect of hair dyes and bleaches on hair proteins [3,4]. To add to such electrophoretic investigations, in this paper we report on the effect of various hair dyes and bleach on the isoelectric focusing (IEF) patterns of the hair proteins. The dyes and bleach used for this study are listed in Table 1. Hair samples (hair shafts from the scalp) without prior hair cosmetic treatment were obtained from 31 healthy Japanese adults. The samples were washed with petroleum ether and ethanol, after which separate samples were treated with one of the listed dyes or the bleach in vitro, according to the instructions for each product. The subsequent extraction of the non-carboxymethylated fibrous proteins from both the treated hair samples and untreated hair samples was performed according to the method of Carracedo et al. [5]. Hair fragments (1 mg) were suspended in 100 pL of 0.05 M Tris-HC1, pH 9.3, containing 8 M urea and 0.05 M dithiothreitol (DTT), for 24 h at room temperature, with occasional shaking. The suspension was then centrifuged at 2500 rpm for 5 min and, to each 5 pLof resulting supernatant, 1 pL of 0.1 M DTT was added at least 10 min before IEF was initiated. Protein concentrations of the supernatant were determined by the method of Lowry et al. [ 6 ] , modified by Ross and Schatz [7], with bovine serum albumin as standard. Correspondence: Dr. Atsushi Nagai, Department of Legal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan Abbreviations: C, relative percentage of cross-linker in a polyacrylamide gel; IEF, isoelectric focusing; T,total monomer concentrations in polyacrylamide gel
0VCH Verlagsgesellschaft m h H , D-6940 Weinheim, 1991
IEF was performed in 0.5 mm thick polyacrylamide slab gels (5.0% T, 2.8% C) with 2.0% Ampholine carrier ampholytes (pH 4.0-6.5) and 4 M urea, using a Multiphorchamber (LKB,Bromma, Sweden) and a power supply, Model 3000/ 300 (Bio-Rad, Richmond, CA). Polymerization was carried out with ammonium persulfate and N,N,N', N' -tetramethylenediamine at room temperature. The electrode solutions were 1 M NaOH for the cathode and 1 M H,PO, for the anode. The gels were prefocused at 2 W constant power for 30 min at a cooling temperature of 4°C. Forty pL samples, without adjusting protein concentrations, were applied to Whatman 3 MM filter papers (10 X 10 mm) at a distance of 2 cm from the cathode and focusing was continued for 1 h. Then the papers were removed and focusing was continued at 4 W (maximum voltage of 1600 V) for an additional 180 min. After IEF, the gels were fixed in 20% trichloroacetic acid for 30 min and rinsed in a destaining solution (50% ethanol and 17% acetic acid) for 30 rnin with shaking. The gels were then stained with Coomassie Brilliant Blue R-250 for 1 h at room temperature and destained until sufficient contrast in the bands was achieved. The pZ values were determined by directly measuring the pH on the surface of the gel using a pH meter (Corning, NY, USA) with a microelectrode (Toko Chemical Industries, Tokyo, Japan). First, the IEF of the extracted proteins from 31 untreated hair specimens revealed that all the IEF patterns were exactly the same. Carracedo et al. [5] have reported electrophoretic variants of human hair proteins without carboxymethylation by IEF and by silver staining. Gerhard [8],Gerhard and Hermes [4], and Schimkat et al. [9] have also reported variants of human hair proteins without carboxymethylation on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Even so, we were unable to find any variants in our specimens on using the present method. In Fig. 1, the IEF results of the extracted proteins from dyed and bleached hair are shown. Note that the protein patterns of hair treated with a temporary dye (color spray) or a semi-permanent dye (acidic color) were identical to the protein patterns of untreated hair (Fig. 1, lanes 1-3). However, the IEF patterns of protein extracts from hair that was treated with a permanent hair dye (metallic, or alkaline oxidative, or acidic oxidative), or with a hair bleach were distinctly different from the pattern of untreated hair proteins. Additionally, the hair protein patterns of hair that was treated with an alkaline oxidative dye were identical to the pattern of the extracted proteins from bleached hair. In both types of patterns, a distinct band was observed at pH 5.2. Moreover, compared with the IEF patterns of the untreated hair proteins, the bands at pH 4.0-4.3 and pH 5.1 0173-0835/91/0606-0451 $3.50+.25/0
452
Electrophoresis 1991, 12, 451-453
A. Nagai et al.
Table 1. List of hair dye and hair bleach uscd for this study Effect on hair Temporary Semi-permanent Permanent
Bleaching
a) b) c) d) e)
Hair dyes and bleach used
Commercial name (Color)
Color spray Acidic color Metallic dye Alkaline oxidative dye Acidic oxidative dye Hair bleach
Ford Color Coat Spray (sepia) Color Story (madder-red) Marron (black) Simone Hair Color (safari brown) Real Care Tone (light brown) Sirnone Bleach
Source
4 b) c) d) el d)
Ford Hair Cosmetics, Osaka, Japan Arimino, Tokyo, Japan Yamahatsu Sangyo, Osaka, Japan Hoyu, Nagoya, Japan Real Chemical, Tokyo, Japan
were more intense, while the band at pH 5.3 was weaker (Fig. 1, lanes 5 and 7). As for hair that was treated with the acidic oxidative dye, the bands at pH 4.8-5.2 were weak (Fig. 1,lane 6).With respect to hairtreated with the metallic
1
2
3
4
5
6
7
Figure 1. IEF patterns of extracted proteins from hair treated with hair dyes or bleach. Lane (1) untreated; (2) color spray-treated; (3) acidic color-treated; (4) metallic dye-treated; (5) alkaline oxidative dye-treated; (6) acidic oxidative dye-treated; and (7) bleach-treated hair.
1
2
3
4
5
6
Figure2. IEFpatterns ofextracted proteins from hair treated with an alkaline agent or an oxidative agent. Lane (l),unreated; (2) 28% ammonium hydroxide-treated; ( 3 ) 6% hydrogen peroxide-treated; (4) with an equivalent mixture of 28% ammonium hydroxide and 6 % hydrogen peroxide; (5) alkaline oxidative dye-treated; and (6) bleach-treated hair.
dye, the band patterns were faint except for the pH 4.0 band andafewweakbandsat pH4.3-4.6(Fig. l,lane4).Changes in the IEF patterns were exactly the same for all the samples of the present study. Average protein concentrations of extracts from treated or untreated hair were as follows: untreated, 6.24 mg/mL; color spray-treated, 6.23 mg/mL; acidic color-treated, 6.41 mg/mL; metallic dye-treated, I .23 mg/mL; alkaline oxidative dye-treated, 9.13 mg/mL; acidic oxidative dye-treated, 6.80 mg/mL; bleach-treated, 8.00 mg/mL. Both alkaline oxidative dyes and hair bleaches contain an alkaline agent (mainly ammonia) and an oxidative agent (mainly hydrogen peroxide) [l].The former agent is used to make the hairs swell for easy permeation of the oxidative pigments and to decompose hydrogen peroxide into oxygen and hydrogen [I], whereas the latter agent is used to convert the aromatic amines in oxidative dyes into oxidized pigments and to decolor the melanin of the hair by its oxygen [l].It is further known that these agents chemically damage the hair proteins [2, 101. Therefore, to investigate whether these agents have an effect on the IEF hair protein patterns, the samples were either treated with 28 O/o ammonium hydroxide, 6% hydrogen peroxide, or an equivalent mixture of these solutions for up to 30 min, following the specific instructions on the use of each dye. The IEF hair protein patterns extracted from these treated hairs are shown in Fig. 2. The IEF hair protein patterns of hair treated with only ammonium hydroxide orwith only hydrogen peroxide were similar to the IEF patterns of the untreated hair proteins (Fig. 2, lanes 1-3). However, the IEF hair protein patterns of hair that was treated with an equivalent mixture of these two chemicales were similar to the IEF hair protein patterns of hair that was treated with an alkaline oxidative dye or a hair bleach (Fig. 2, lanes 4-6). These results indicate that the changes in the IEF hair protein patterns, observed after treatment with an alkaline oxidative dye or a hair bleach, were caused by a combined effect of an alkaline agent and an oxidative agent. The weak IEF patterns of hair protein treated with a metallic dye result from a distinctly lower protein concentration of the extracts. The concentration of about one-fifth of that from untreated hair is probably due to some components of the metallic dye, decreasing protein solubility. The cause for the change in the IEF pattern in hair treated with an acidic oxidative dye remains to be elucidated. Some reports state that hair cosmetic treatment, i.e., hair dying, bleaching and/or permanent waving, obstruct the detection of electrophoretic variants of the hair proteins [3,4].
Electrophoresis 1991, 12, 453-456
Quantification of proteins by colloidal silver
In the present study, we were able to demonstrate that metallic dying, oxidative dying, and bleaching induce changes in the IEF patterns and in the intensity of the hair protein bands. Furthermore, these results indicate that hair treated with a permanent hair dye and hair that is bleached can be differentiated from untreated hair by its characteristic IEF patterns and that metallic dyed hair as well as acidic oxidative dyed hair can be distinguished from hair that has been treated with other dyes. It was not clarified whether these effects accumulative with prolonged use of hair dyes or a hair bleach, are reversible following cessation, or complementary when these products are used in sequence. These results may prove useful for forensic cases requiring hair identification. This work was supported in part by a Scientific Research Grant (No. 01 770383)from the Ministry of Education, Science, and Culture, Japan. Received December 25, 1990
Pavel Draber Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague
453
References [ l ] Watanabe, Y., Tamura, T., Harada, H. and Yagihara, Y., Hairscience (in Japanese), Japanese SocietyofHairScience,Tokyo 1986,pp. 117154. [Z] Watanabe, Y., Tamura, T., Harada, H. and Yagihara, Y., Hair Science (in Japanese), Japanese Society of Hair Science,Tokyo 1986, pp. 95116. 131 Zahn, H. G., Bindewald, I. and Marshall, R. C.,J. For. Sci. SOC.1984, 24, 432. [4] Gerhard, M. and Hermes, M., Electrophoresis 1987. 8, 490-492. [S] Carracedo, A. Concheiro, L. and Requena, I., For. Sci. Int. 1985, 29, 83-89. [6] Lowry, 0. H., Rosebrough,N. J., Farr,A. L. and Randall, R. J.,J.B i d . Chem. 1951, 193, 265-275. [7] Ross, E. and Schatz, G., Anal. Biochem. 1973, 54, 304-306. 181 Gerhard, M., Electrophoresis 1987, 8, 153-157. [9] Schimkat, M., Baur, M. P. and Henke, J., Hum. Genet. 1990,85,311314. [lo] Toda,J.andOkamoto,K.,in:Arao,T.,Takashima,I.,Toda,J.,Hori,Y. and Okamoto, K. (Eds.), Medicine of Hair (in Japanese) Bunkodo, Tokyo 1987, pp. 280-296.
Quantification of proteins in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using colloidal silver A rapid and simple assay for quantification of proteins in sodium dodecyl sulfatesample buffer is described. Proteins are bound to a nitrocellulose membrane, stained with colloidal silver, and quantified by transmission densitometry. The staining requires 1 pL of protein sample and takes less than 20 min. Good linearity between the staining intensityand amount ofproteins is in the range of5-100 ng.
A quantitative analysis of proteins separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis often requires measurement of protein concentration in the SDS-sample buffer. When only a limited amount of material is available, a sensitive method is necessary for protein quantification. The use of nitrocellulose (NC) membranes for immobilization of macromolecules is well documented; bound polypeptides/proteins can be stained and quantified by densitometry [l-61. Silver staining [2] or AuroDye staining [3,5] of NC membrane makes it possible to detect protein quantities in the nanogram range. However, staining with AuroDye, although simple, requires several hours, and silver staining, based on a combination of silver photodevelopment and chemical development, is a multi~
~
Correspondence: Dr. Pavel Driber, Institute of Molecular Genetics, Czechoslovak Academy of Sciences,Videfiska 1083,CS-142 20 Prague 4, Czechoslovakia
Abbreviations: BSA, bovine serum albumin; NC, nitrocellulose; SDS, sodium dodecyl sulfate
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1991
step procedure. It has been shown that proteins electrophoretically transferred onto NC membranes could also be detected with high sensitivity by staining with colloidal silver [7]. This paper describes a simple and rapid quantification of proteins in SDS-sample buffer [8] by spotting the samples onto NC membranes and staining the dots with colloidal silver. NC membranes BA 85, BAS 85 (Schleicher and Schuell, Dassel, Germany), Synpor 6 (Synthesia; Prague, Czechoslovakia) or MSI NC membrane (MSI, Westboro, MA) with 0.45 V r n pores were used. All substances were of analytical grade and were obtained from Serva Feinbiochemica (Heidelberg, Germany) or Lachema (Brno, Czechoslovakia). U1trapure water from a Millipore water purification system was used throughout the assay. Bovine serum albumin (BSA, Cat. No. 11923) and lysozyme from chicken egg white (Cat. No. 28260) were supplied by Serva, bovine y-globulin (Cat. No. G SOOS), chicken egg albumin (Cat. No. A 5503), a-chymotrypsin from bovine pancreas (Cat. No. C 3142), aprotinin from bovine lung (Cat. No. A 1153), 0173-0835/91/0606-0453 $3.50+.25/0
