Use of thioredoxin, thioredoxin-derived, or thioredoxin-like dithiol peptides in hair care preparations

The subject invention enables a more efficient management of hair by providing a novel preparation for waving, straightening, softening, or removing hair, employing as a key ingredient the compound thioredoxin or a thioredoxin-derived, or thioredoxin-like, dithiol peptide in combination with a sulfite or bisulfite compound.

BACKGROUND OF THE INVENTION 
The care of hair has been of utmost importance to mankind from the 
beginning of recorded history. The reign of Queen Elizabeth (1558-1603) 
became noted for its attention to the finer aspects of hair styling; it 
was Her Majesty who set standard. During this Elizabethan period, hair was 
arranged in elaborate high coiffures, and curled and frizzed by whatever 
means were available. Needless to say, as measured by present day 
available hair care products and methods, the Elizabethan hair care 
procedures were primitive, at best. The discovery of new chemicals and 
properties thereof led to the beginning of hair care products designed to 
beautify and maintain the hair in healthy, youthful state. These desirable 
human hair properties were achieved by use of a variety of hair care 
products, including hair dyes and products used to impart a wave to the 
hair. Wavy hair is considered a desirable human hair feature, whereas 
straight hair is usually held in less favor. Because of these human 
demands to beautify the hair, there has evolved a multitude of hair care 
products with a variety of claims and promises. With hair care products 
designed to dye or wave the hair, it has been found that the structure of 
the hair shaft itself must be reckoned with in order to have a product 
which would give the desired results. A key detail of the hair shaft, 
which is predominantly keratinaceous in nature, is that the keratin fibers 
are bonded together by disulfide crosslinkages. It is this detail of the 
hair structure which the subject invention is concerned with. The prior 
art discloses the severance of the disulfide crosslink with, enter alia, 
various chemical agents. 
In a normal `tepid` waving process, keratin disulfides are reduced by use 
of either sulfites or bisulfites. Sulfite and bisulfite waving have the 
advantage of being less damaging than a thioglycolate wave with less 
likelihood of overprocessing. Drawbacks to the sulfite and bisulfite 
waving process however, are that they give soft waves and the permanent 
does not last as long as the bisulfite wave is the formation of keratin 
thiosulfates, thioglycolate wave. An additional detraction of the commonly 
known as Bunte salts. The presence of residual Bunte salts can lead to 
relaxation of the curl through disulfide exchange and to lanthionine 
formation causing irreversible hair damage. These Bunte salts can also 
affect the texture and feel of hair. 
BRIEF SUMMARY OF THE INVENTION 
The subject invention concerns the surprising and advantageous discovery 
that the use of sulfite or bisulfite compounds in hair care preparations 
can be dramatically improved upon by use of thioredoxin or a 
thioredoxin-derived, or thioredoxin-like, dithiol peptide compounds. This 
combination gives rise to a synergistic effect in terms of efficiently 
breaking the disulfide bond of hair keratin. The net result is that 
significantly lesser amounts of sulfite or bisulfite compounds are needed 
to produce the desired effect in the hair. Coupled with this reduction of 
sulfite or bisulfite compounds use are other desirable features: The hair 
can be waved in a shorter time and Bunte salts formed can be cleaved from 
the hair fiber. In achieving these desirable results, the subject 
invention enhances rather than compromises the reductive properties of 
sulfite or bisulfite compounds and additive dithiol peptides. 
DETAILED DISCLOSURE OF THE INVENTION 
Upon adding thioredoxin or a thioredoxin-derived, or thioredoxin-like, 
dithiol peptide to a hair care product containing a bisulfite or sulfite 
compound, e.g., a hair preparation for straightening, waving, removing, or 
softening hair, there is realized a synergistic effect whereby 
significantly lesser amounts of sulfite or bisulfite compound are needed 
to produce the desired effect. For example, in commercial practice, sodium 
or ammonium bisulfite is used in waving locations at about 7.0% 
concentration levels. As the bisulfite concentration increases from about 
0.01% to about 7.0%, the amount of hair curling increases with a leveling 
off occurring at about 7.0%. By adding thioredoxin or a 
thioredoxin-derived, or thioredoxin-like, dithiol peptide to the waving 
preparation, the bisulfite concentration can be reduced by a factor of two 
to about 3.2% and still give the same amount of waving as a commercial 
waving lotion containing 7.0% bisulfite. 
The concentration of thioredoxin or one of the thioredoxin-derived, or 
thioredoxin-like, dithiol peptides which can be used to enhance the effect 
of a sulfite or bisulfite compound ranges from about 1 to about 100 
nmole/ml. The optimal concentration for intact bacterial thioredoxin 
appears to be about 2 nmole/ml. It should be recognized that the precise 
level of thioredoxin or thioredoxin-derived, or thioredoxin-like, dithiol 
peptide in combination with a sulfite of bisulfite compound can be readily 
ascertained for a particular hair sample by a person skilled in the hair 
care art having possession of the subject invention. 
Thioredoxins are low molecular weight dithiol proteins that have the 
ability to reduce disulfides in typical organic compounds such as Ellman's 
reagent or disulfides as they exist naturally in a variety of proteins 
(Holmgren, A. [1981] Trends in Biochemical Science 6:26-39). 
Thioredoxin and thioredoxin-derived, or thioredoxin-like, dithiol peptides 
within the scope of the subject invention are exemplified by the following 
compounds: 
(1) thioredoxin isolated from Escherichia coli (Laurent, T. C., Moore, E. 
C., and Reinchard, P. [1964] J. Biol. Chem. 239:3436-3445); 
(2) thioredoxins isolated from other sources, e.g., thioredoxin isolated 
from yeast (Porque, G. P., Baldesten, A., and Reichard, P. [1970] J. Biol. 
Chem. 245:2363-2379); Cyanobacterium (Gleason, F.K. and Holmgren, A. 
[1983] in "Thioredoxins, Structure and Function" [P. Gadal, ed.] Editions 
du Centre National de la Recherche Scientifique); rate (Guerara, J., 
Moore, E. C., and Ward, D. NM. [1983] ibid); T.sub.4 bacteriophage 
(Soderberg, B.-O., Sjoberg, B.-M., Sonnerstam, U., and Braden, C.-I. 
[1978] Proc. Natl. Acad. Sci. U.S.A. 75:5827-5830); purification of 
mammalian thioredoxin 
(Luthman, M. and Holmgren, A. [1982] Biochem. 121:6628-6633); further, 
thioredoxin from a human source can be used in the subject invention; 
(3) thioredoxin-derived dithiol peptides representing peptides produced by 
cleavage of intact thioredoxins, as described infra. One such example of 
this class of thioredoxin-derived peptides is the fragment containing 
residues 1 through 37 (i.e., T.sub.1-37) produced by cyanogen bromide 
cleavage of thioredoxin from E. coli. The important feature of these 
thioredoxin-derived dithiol peptides is that they contain the redox-active 
peptides sequence, Cys-X-Y-Cys, wherein X and Y, independently, can be any 
of the natural 20 amino acids. For example, the redoxactive peptide 
sequence from E. coli thioredoxin is Cys-Gly-Pro-Cys (Cys=cysteine, 
Gly=glycine, Pro=proline). Also the redox-active sequences Cys-X-Cys-Lys 
or Trp-Cys-X-Y-Cys-Lys, wherein X and Y are as defined above, for example, 
Cys-Gly-Pro-Cys-Lys or Trp-Cys-Gly-Pro-Cys-Lys can be used; and 
(4) thioredoxin-like dithiol peptides that inter alia have the intrinsic 
ability to catalyze the reduction of protein disulfides. These 
thioredoxon-like dithiol peptides will generally have the characteristic 
of containing a pair of cysteine residues which form a redox-active 
disulfide. This example includes peptides, derived from natural sources or 
constructed synthetically, that include the same redox-active sequence as 
disclosed above, for example in E. coli thioredoxin, Cys-Gly-Pro-Cys, 
Cys-Gly-Pro-Cys-Lys, or Trp-Cys-Gly-Pro-Cys-Lys, or analogous sequences 
from other thioredoxins such as that encoded for by T4 bacteriophage, 
Cys-Val-Tyr-Cys (Cys=cysteine, Val=valine, Tyr=tyrosine) Soderberg, B.-O., 
Sjoberg, B.-M., Sonnerstam, U., and Branden, C.-I. [1978] Proc. Natl. 
Acad. Sci. U.S.A. 75:5827-5830). Other thioredoxin-like peptides include 
the class of seed proteins called purothionins that have intrinsic 
thioredoxin-like activity (Wada, K. and Buchanan, B.B. [1983] in 
"Thioredoxins, Structure and Function" [Gadal, P., ed.] Editions du Centre 
National de la Recherche Scientifique). 
Following are examples which illustrate products of the invention and 
procedures, including the best mode, for practicing the invention. These 
examples should not be construed as limiting. All percentages are by 
weight and all solvent mixture proportions are by volume unless otherwise 
noted.

EXAMPLE 1 BISULFITE WAVING SOLUTIONS 
The bisulfite waving solution consisted of 7% (w/w) ammonium bisulfite, 
4.65% (w/w) ethanol, and 0.6% (w/w) polyoxyethylene(23) lauryl ether. The 
pH was adjusted to 7.5 with ammonium hydroxide. All dilutions of the 7% 
solution were made using a diluent consisting of all components except the 
ammonium bisulfite. The neutralizer contained 2.3% hydrogen peroxide 
adjusted to pH 3.3 with dilute phosphoric acid. All solutions were 
overlayed with argon or nitrogen and stored in the dark at room 
temperature for up to 2 months. 
Hair Waving Assay: The influence of thioredoxin and active site peptides on 
bisulfite permanent waving was determined by direct hair waving assays. 
Tresses were divided into smaller tresses approximately 0.5 cm wide and 12 
to 13 cm in length. Each tress weighed approximately 0.2 g. 
Intact tresses were shampooed before waving with SILKIENCE.TM. for normal 
hair (The Gillette Company, Boston, Mass.). Each tress was shampooed and 
thoroughly rinsed two times before being combed through and allowed to air 
dry. Bleached-waved tresses were used as received with no additional 
treatment. 
Each tress was treated with a total of 3 ml of waving lotion. Half the 
lotion (1.5 ml) was applied to the tress and the saturated tress remained 
at room temperature for 20 minutes before being combed through and rolled. 
An end paper was folded around the tress to assure that the tress was flat 
and all ends were covered. The tress was rolled firmly and evenly on the 
medium orange rods (0.6-0.7 mm diameter) available in the RAVE.TM. curler 
assortment (Chesebrough-Pond's Inc., Trumbull, Conn.). After the tress was 
rolled it remained at room temperature for an additional 20 minutes. The 
tress was then saturated with the remaining waving lotion, covered with 
plastic wrap, and incubated for 60 minutes at 33-34.degree. C. After 60 
minutes the tress was rinsed for 3 minutes with 40.degree. C. tap water, 
blotted dry, and neutralized. 
The tress was saturated with neutralizer and resaturated after 90 seconds. 
After 10 minutes at room temperature, the rod was removed from the tress 
and the neutralizer worked down to the end of the tress. After an 
additional 2 minutes the tress was rinsed for 2 minutes with 40.degree. C. 
tap water and the hanging length was measured immediately while the tress 
was wet. 
Differences in the amount of waving from the different solutions were 
quantitated by measuring the hanging length before and after waving. The 
relative hair length was calculated according to the following equation: 
EQU RHL=L.sub.a /L.sub.b 
where RHL is the relative hair length, L.sub.b the length before waving, 
and L.sub.a the length after waving. 
EXAMPLE 2 Influence of Thioredoxin 
Thioredoxin was effective in increasing the waving obtained from a 
bisulfite solution in hair. As anticipated, the amount of waving with 
bisulfite alone was significantly less in intact hair (RHL=0.74) as 
compared to bleached-waved hair (RHL=0.70). The increase in waving with 
the addition of thioredoxin was also more significant. The RHL with the 
addition of thioredoxin was 0.69 for intact hair compared to 0.68 for 
bleached-waved hair. Thioredoxin in 3.2% bisulfite produced more waving 
than a 7% commercial preparation (CLAIROL KINDNESS.TM.). The amount of 
dithiol required to give the maximal effect was 2-5 .mu.M. 
The addition of thioredoxin affected the processing time required in 
treated hair. The presence of thioredoxin significantly reduces the 
observed biphasic nature of the reaction. For example, in intact hair, 
thioredoxin increased the amount of waving at a reaction time as short as 
15 minutes. Intact tresses were waved with a 3.2% bisulfite solution alone 
or containing 5 .mu.M thioredoxin. As with treated hair, the greatest 
increase in waving with the addition of thioredoxin was at 45 minutes. 
These results indicate two important conclusions. First, with the addition 
of thioredoxin the amount of waving in a bisulfite system can be 
increased. Second, if no increase in the amount of waving is desired, the 
processing time can be reduced from 60 minutes to 40-45 minutes with the 
addition of thioredoxin to a 3.2% bisulfite solution. 
EXAMPLE 3 INFLUENCE OF THIOREDOXIN AND CYSTEIN METHYL ESTER 
Studies on the cleavage of a model Bunte salt with thioredoxin supported 
the idea that at low pH a stable sulfonated thioredoxin intermediate is 
formed and this intermediate could be recycled to reduced thioredoxin by 
the addition of a secondary reductant such as cysteine methyl ester or 
cysteine. Above neutral pH this intermediate is unstable and oxidized 
thioredoxin is formed. 
The effect of reduced thioredoxin (5 .mu.M) and various concentrations of 
cysteine methyl ester on permanent waving at neutral pH was determined. 
Cysteine methyl ester in the absence of thioredoxin increased the amount 
of waving though it was not as effective as intact thioredoxin. The 
presence of cysteine methyl ester had no effect on waving with intact 
thioredoxin. 
Waving obtained with reduced thioredoxin in the absence of cysteine methyl 
ester was comparable to that using oxidized thioredoxin. Since the reduced 
form of the enzyme is necessary to cleave Bunte salts, this shows that 
thioredoxin may be involved in something other that Bunte salt cleavage 
when added directly to the bisulfite waving solution. The other 
possibility is that thioredoxin is reduced by the bisulfite and then 
cleaves Bunte salts. 
EXAMPLE 4 INFLUENCE OF T.sub.31-36 AND CYSTEINE 
The effect of a secondary reductant with T.sub.31-36 
(trp-Cys-Gly-Pro-Cys-Lys) was studied to determined whether the amount of 
waving obtained with the minimal peptide could be increased. T.sub.31-36 
is one of the minimal active site peptides and is not limited by molecular 
size as is intact thioredoxin. Cysteine was used as the secondary 
reductant in the presence of reduced T.sub.31-36 (2 .mu.M) and 3.5% 
bisulfite. Cysteine itself gave increased waving in the .mu.M range. 
However, the presence of both T.sub.31-36 and cysteine (10 .mu.M) in 
bisulfite showed no increase in waving as compared to bisulfite alone. 
Preparation of Thioredoxin Compounds 
Production of Purified Thioredoxin: Thioredoxin is purified either from a 
commercial source of E. coli, strain B (Grain Processing Corp., 
Minneapolis, Minn.) or from any of a number of common strains of E. coli 
grown by standard procedures (Pigiet, V. and Conley, R. R. [1977] J. Biol 
Chem. 252:6367-6372). The protein is purified using standard procedures 
including chromatography on ion exchange and molecular sieve columns 
(Williams, C. H., Zanetti, G., Arscott, L. D., and McAllister, J. K. 
[1967] J. Biol. Chem. 242:5226-5231; and McEvoy, M., Lantz, C., Lunn, C. 
A., and Pigiet, V. [1981] J. Biol Chem. 256:6646-6650). Thioredoxin was at 
least 95% homogeneous as determined by SDS-polyacrylamide gel 
electrophoresis. The enzyme was stored in 5 ml aliquots in -20.degree. C. 
in 0.5M Tris, pH 7.4 with 1 mM EDTA. 
Assay for thioredoxin: Thioredoxin protein was assayed immunologically 
using quantitative rocket immunoelectrophoresis as described previously 
(McEvoy, M., Lantz, C., Lunn, C. A., and Pigiet, V. [1981] J. Biol. Chem. 
256:6646-6650). 
Isolation of T.sub.1-37 : T.sub.1-37 was produced by cyanogen bromide 
cleavage of thioredoxin. A sample of thioredoxin was dialyzed in water for 
12 hr. at 4.degree. C. The sample was taken to dryness and resuspended in 
70% formic acid and added to thioredoxin in a 50-fold molar excess. The 
solution was purged with nitrogen and incubated at room temperature in the 
dark for at least 24 hr. At the completion of the cleavage reaction the 
solution was dried under nitrogen and resuspended in a minimal amount of 
concentrated ammonium hydroxide. When the sample was dissolved, the pH of 
the sample was adjusted to 8.0 with concentrated HCl. The sample was 
stored at -20.degree. C. under argon and aliquots were removed for 
purification. 
T.sub.1-37 was isolated by affinity chromatography on thiopropyl sepharose 
6B. A sample of the CNBr digest was incubated with a 2-fold molar excess 
of DTT for 10 minutes at room temperature before being applied to a 
thiopropyl column equilibrated with 0.1M Tris, pH 7.5, containing 0.5M 
NaCl and 1 mM EDTA. The column was washed with two column volumes of the 
equilibrating buffer containing 2M urea to remove any T.sub.38-108 that 
was non-specifically bound. The column was then washed with an additional 
2 column volumes of equilibrating buffer. T.sub.1-37 was eluted from the 
column with 25mM DTT in equilibrating buffer. The sample was analyzed for 
homogeneity by reverse phase high pressure liquid chromatography on a 
Waters .mu.-Bondpak C.sub.18 column attached to a Beckman Model 421 system 
monitored at 214 nm. A 0-60% gradient of acetonitrile containing 0.08 to 
0.1% TFA was used to elute the peptide at a flow rate of 2 ml/min. The 
peptide was judged to be greater than 95% pure by this procedure. 
DTT was removed from the sample by exclusion chromatography. The sample 
volume was reduced using a Savant Speed Vac Concentrator and applied to a 
1 cm.times.24 cm column of Sephadex.TM. G-25-40 equilibrated with 0.05M 
Tris, 1 mM EDTA, pH 7.4 (TE buffer). The 0.3 ml fractions collected were 
monitored at 280 nm. The samples containing t.sub.1-37 were pooled and the 
concentration determined by A.sub.280 (.sub.280 =10,000 cm.sup.-1 
M.sup.-1). The sample was immediately used in the waving assay. 
Isolation of T.sub.32-37 : T.sub.32-37 was isolated from a chymotryptic 
digest of the CNBr digest. Chymotrypsin was added to a prewarmed (37+ C.) 
solution of CMBr digest to a final concentration of 1:20 (w/w) 
chymotrypsin to peptide. After incubating the sample for 1 hr 
L-1-Tosylamide-2-phenylethylchloromethyl ketone (TPCK) was added in a 1:1 
molar ratio to chymotrypsin to stop the reaction. 
The sample was loaded onto a Waters .mu.-Bondpak C.sub.18 column attached 
to a Beckman Model 421 system monitored at 214 nm. The solvent system 
employed was 0.1% trifluoroacetic acid (Buffer A) and 0.08% 
trifluoroacetic acid in acetonitrile (Buffer B). A gradient from 0-30% B 
over 30 minutes and 30-60% over 15 minutes was used to separate the 
peptides at a flow rate of 2 ml/min. The peak identified as T.sub.32-37 
was collected, taken to dryness, and stored under argon at -20.degree. C. 
Reduction of T.sub.31-36 : T.sub.31-36 was obtained synthetically and was 
reduced for several experiments. The peptide was incubated at 37.degree. 
C. for 1 hr with a 5-fold molar excess of DTT. The DTT was removed by HPLC 
as described for the isolation of T.sub.32-37. T.sub.31-36 was taken to 
dryness, reconstituted with a minimal volume of TE buffer and used 
immediately in the waving assay. The procedure was only partially 
effective, with only 30% of the peptide reduced as determined by cleavage 
of a model Bunte salt.