The Effect of Reducing Agents

Transcription

1 I. Soc. Cosmet. Chem., 22, (August 18, 1971) on The Effect of Reducing Agents Fingernail Keratin NANCY F. WOLEJSZA, B.A.,* STANLEY G. ELFBAUM, Ph.D.,* and MARIA A. WOLFRAM, Ph.D.* Synopsis--The CYSTINE crosslinks in human FINGERNAILS have been subjected to RE- DUCTION by AMMONIUM BISULFITE, DITHIOTHREITOL, AMMONIUM THIO- GLYCOLATE, and TETRAKIS(HYDROXYMETHYL)PHOSPHONIUM CHLORIDE. Reduced fingernail KERATIN exhibits increased equilibrium liquid retention properties and an enhanced methyl orange uptake. The reduction process is ahnost completely reversible when dithiothreitol or ammonium bisulfite is used as the reductant, and only partially reversible when ammonium thioglycolate or tetrakis(hydroxymethyl)phosphonium chloride is used. Explanations are offered for the partial reversibility of the last two processes. INTRODUCTION Extensive work has been done on the reduction of cystine in keratin substrates, primarily wool and hair (1-6). Amino acid analyses both in this laboratory (Table I) and in others (7, 8) have shown that the amount of cystine in fingernails is appreciable. This property of fingernails suggested to us that, as in the case of wool and hair, partial or complete reduction of the structural cystine should result in an "opening up" of the keratin structure, and this in turn should lead to an enhanced internal diffusibility and subsequent binding of water or other materials. The present paper investigates several reagents for their effectiveness as fingernail reductants. The reagents selected were ammonium Gillette Company Research Institute, 1413 Research Blvd., Rockville, Md Present address, Boston Medical Laboratory, 19 Bay State Road, Boston, Mass

2 572.JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table Amino Acid Analysis of Adult Fingernails Amino Acid #moles/g g/100 g Lysine Histidine Ammonia Arginine CySO2H Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine I thioglycolate (2), ammonium bisulfite (3), 1,4-dithiothreitol (Cleland's Reagent) (4, 9), and tetrakis(hydroxymethyl)phosphonium chloride (THPC) (6). Equilibrium liquid retention studies (10) and titration by a mercurial (5, 11) were utilized to determine the extent of reduction and of reoxidation by hydrogen peroxide. MATERIALS AND METHODS Pooled fingernail clippings were obtained from three volunteers. After being washed for 5 rain with soap and water and subsequently rinsed with ethanol, the clippings were air-dried and stored at 65% RH and room temperature until use. Analytical grade reagents and solvents were used without further purification. Ammonium thioglycolate (0.1M) was prepared by adjusting thioglycolic acid to ph 9 with concentrated ammonia. Ammonium chloride (0.1M) was adjusted to ph 9 with concentrated ammonia solution. Tetrakis(hydroxymethyl)phosphonium chloride (THPC),* supplied as an 80% solution, was used without further purification. Hooker Chemical Corp., Niagara Falls, N.Y.

3 REDUCING AGENTS AND FINGERNAIL KERATIN 573 To 10-mg samples of clippings, 10 ml of reducing agent was added. Reduction was carried out for different time intervals at 35øC except in the case of THPC, where room temperature was employed. After incubation in the reducing solution, the nails were rinsed for 2-3 seconds in 0.1N acid to retard cysteine reoxidation. Untreated nails rinsed in a similar manner with dilute acid underwent no weight changes. Equilibrium liquid retentions were determined by a technique similar to that described for use on hair by Valko and Barnett (10). Following reduction, the fingernail clippings were soaked for 30 min in a ph 7 buffer solution containing 0.1% Triton X-100 and then centrifuged for 10 min (about 1000 g) on wire screens in securely capped centrifuge tubes. The nails were quickly transferred to capped tared containers and weighed. Dry weights were obtained by drying the nails slowly (about 16 hours) at 80øC to constant weight. Higher drying temperatures were avoided in an attempt to minimize the risk of damage. The amount of liquid retained by the nails was expressed as a per cent of the dry weight. Calculations on selected samples show the standard error to be of the order of 7%. Fingernails incubated in 0.1M ammonium chloride (ph 9) for times up to 2 hours had equilibrium retention values which were identical to those of the untreated controls. Since the actual reductions were carried out at ph 9 or lower it was assumed that the observed changes in equilibrium retention were a result of extent of reduction and not due to structural damage caused by incubation in alkali media. Reoxidation of the reduced nails was carried out by treatment with 3% hydrogen peroxide (self ph) for 15 min at 35øC. The nails were then rinsed with distilled water and subjected to equilibrium liquid re- tention measurements. Binding of methyl orange by reduced fingernails was determined by the dye depletion technique. Samples (10 mg each) of fingernails were immersed in 5 ml of a dilute solution of methyl orange (0.71 X 10-4 or 1.0 X 10-4M) and incubated overnight at ph 2.5 or ph 4.0. The depletion of methyl orange after 18 hours in the dye bath was followed spectrophotometrically by measuring the decrease in optical density at 505 m (ph 2.5) or 476 m (ph 4.0). Values of 1.89 X 104M - and 1.47 X 104M - for the molar extinction coefficients of methyl orange at ph's 2.5 and 4.0, respectively, determined in the laboratory were used. The sulfhydryl content of fingernails was determined by titration with salyrganic acid using a sodium nitroprusside indicator (5, 11). Samples (45 mg) of fingernails were hydrolyzed for 3 - hours with 2.5 ml

4 574 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Of 30% H2SO4 (v/v). After cooling, the solution was diluted to 10 ml with distilled water. Five-milliliter aliquots of the hydrolyzate were made alkaline with saturated sodium carbonate and titrated with salyrganic acid using 1% sodium nitroprusside indicator. The amino acid analysis of the nails was determined by digesting approximately 10 mg in 6N HC1 for 24 hours at 105ø-110øC. After the hydrolysis was completed, the hydrolyzate was filtered and reduced to dryness by freeze drying and then transferred to a 25-cc volumetric flask and diluted with ph 2.2 sodium citrate buffer. Aliquots were then applied to the columns of the Amino Acid Analyzer. RESULTS With 0.1M dithiothreitol (ph 4.6), 0.1M ammonium thioglycolate (ph 9.0), and 1M ammonium bisulfite (ph 6.0), the maximum increase in liquid retention was achieved within 2 hours at 35øC (Figs. 1-3). Beyond this time additional significant increases in swelling were not observed.,-. 7O ß Time (min) Figure 1. Reduction of human fingernails with dithiothreitol X Untreated fingernails ß Reduced fingernails 0 Fingernails after reoxidatizn With 1M THPC (ph 5.3), substantial swelling was not observed until 22 hours of exposure at room temperature had elapsed (Fig. 4). Observations at longer treatment times through 67 hours continued to show increased levels of liquid retention. Model M 7800, Phoenix Precision Instrument Co., Philadelphia, Pa.

5 REDUCING AGENTS AND FINGERNAIL KERATIN ) I 120 Time (min) Figure 2. Reduction of human fingernails with ammonium thioglycolate X Untreated fingernails ß Reduced fingernails O Fingernails after reoxidation õ so Time (min) Figure 3. Reduction of human fingernails with ammonium bisulfite X Untreated fingernails ß Reduced fingernails O Fingernails after reoxidation The increase in sulfhydryl content which should accompany reduction was demonstrated by titration of samples of reduced fingernails (ammonium bisulfite, 90 min, 35øC) with mercurial salyrganic acid (Table II). The enhanced swelling induced by treatment with dithiothreitol and ammonium bisulfite was eliminated by reoxidation ot the sulfhydryl

6 576 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 70 ß 3 ) 60 Time (hrs) Figure d. Reduction of human fingernails with THPC X Untreated fingernails ß Reduced fingernails O Fingernails after reoxidation Table Sulfhydryl Content of Reduced Fingernails II meq SH/g nails Untreated control fingernails Reduced fingernails a Reduced with 1M ammonium bisulfite for 90 min at 35 øc. groups with hydrogen peroxide (Figs. 1 and 3). The equilibrium liquid retention properties of the reduced and then reoxidized nails were approximately the same as those of the untreated controls. With nails reduced by ammonium thioglycolate (Fig. 2) or by THPC (Fig. 4), only partial reversal was realized. In addition to increases sulfhydryl content and water-binding (or liquid retention), reduced nails should show an increased uptake of other substances. Initial investigations have borne this out. The uptake of methyl orange from an acidic solution (ph 2.5 or 4.0) was greater for reduced nails (ammonium bisulfite, 15 or 90 min, 35øC) than for untreated controls (Table III). DISCUSSION The reduction of the cystine crosslinks in fingernails has been achieved with several different materials. In all cases but one, the time

7 REDUCING AGENTS AND FiNGERNAIl, KERATIN 577 Table I I [ Uptakc of Methyl Orangc by Fingernails Moles dye bound/rag nail (X 10-7) Untreatcd Reduced Methyl orange (0.71 X 10-4M) in 0.01N H. SO4 (ph 2.5) ' Methyl orange (1.0 X 10-aM) buffer (ph 4.0) b Fingernails previously reduced for 15 min by ammonium bisulfite at 35 øc. Fingernails previously reduced for 90 min by ammonium bisulfite at 35øC. required to increase the liquid retention o[ the nails appreciably was relatively short, less than 2 hours. With THPG at room temperature, a much longer time was required. The sulfhydryl content o[ fingernails was shown to increase upon reduction. The extent o[ reduction was demonstrated by increased equilibrium liquid retention properties and by an increased methyl orange uptake. Nails reduced with ammonium bisulfite and dithiothreitol have been almost completely reoxidized to their original states, as judged by liquid retention data. With ammonium thioglycol,re as the reductant, the reversal process was partial. Some irreversible structural de[onnity may have occurred in our work, as the reduction process was carried out at alkaline conditions. With THPG, the very long incubation periods required [or substantial reduction may also allow [or some irreversible structural damage to occur. REFERENCES (Received January 18, 1971) (1) Alexander, P., Wool, Its Chemistry aud Phy,ics, Chapman and Hall, London. England, 1954, p (2) Patterson, W. I., Geiger, W. (;., Mizell, L. P., and Harris, M., Role of cysline in the structure of the fibrous protein, wool,.l. Re,. Nat. Bur. Stand., 27, 89 (1941). (3) Speakman, J. B., The reactivity of the sulfur linkage in animal fibers. I. The chemical mechanism of permanent set, J. Soc. Dyer, Colour., 52, 335 (1936). (4) Weigmann, H. D., Reduction of disulfide bonds in keratin with 1,4-dithiothrcitol. I. Kinetic investigation, J. Polym. Sci., Part A-l, õ, 2237 (1968). (5) Wolfram, L. J., and Underwood, D. L., The equilibrium between the disulfide linkage in hair keratin and sulfite or mercaptan, Text. Res. J., $õ, 947 (1966). (6) Jenkins, A.D., and Wolfram, L. J., The chemistry of the reaction between tetrakis (hydroxymethyl) phosphonium chloride and kcratin,.1. Soc. Dyers Colour., 79, 55 (1962).

8 578.JOURNAL OF THE SOCIETY OF COSMETIC (;HENlISTS (7) Ogura, R., Knox, J. M., Griffin, A. C., and Kusuhara, M., The concentration of sulfhydryl and disulfide in human epidermis, hair and nail, J. Invest. Dermatol., 38, 69 (1962). (8) Lustig, B, Katchen, B., and Reiss, F., The amino acid composition of the horny layer of of the human skin, Ibid., 30, 159 (1958). (9) Cleland, W. W., Dithiothreitol, a new protective reagent for SH groups, Biochemistry, 3, 480 (1964). (10) Valko, E. I., and Barnett, G., A study of thc swelling of hair in mixed aqueous solvent, ]. Soc. Cosmet. Chem., 3, 108 (1952). (11) Klotz, I. M., and Carver, B. R., A spectrophotometric titration for the determination of sulfhydryl groups,/trch. Biochem. Biophys., 95, 540 (1961).