METIN AND METACTOMYOSIN*

BY A. SZENT-GY~ROYIAND B. KAMINER

INSTITUTE FOR MUSCLE RESEABCH, MARINE BIOLOGICAL LABORATORY, WOODS HOLE,
                         MASSACHUSETIB

Communicated October 9, 1963

Calcium chloride, if injected into the muscle fiber in minimal quantity, causes an
immediate local contraction. Since this initial observation of Heilbrunn and
Wiercinski,l there has been increasing evidence that Ca++ is directly involved in
the contraction of actomyosin.a  This is rather surprising, for the Ca ion, in general,
is not known to have an affinity for hydrophilous proteins.  Such an affinity could,
however, be expected in colloids having numerous negative groups like SOa, POS, or
COOH.  Since macromolecules carrying such groups may show metachromasia,a
it appeared worthwhile to seek for a metachromatic macromolecule in muscle.

Fresh, washed or glycerinated muscle, on staining with toluidine blue, turns
purple and is thus metachromatic.  Microscopically, the fibers appear to be
diffusely metachromatic.  After disintegrating muscle in a Waring blendor, the
myofibrils, separated by low-speed centrifugation, stain metachromatically, whereas
the free granules and supernatant are orthochromatic.  Thus, the metachromasia
seems to be part of the contractile component of muscle.

If freshly minced muscle of a rabbit is repeatedly washed in cold water or ex-
tracted in 50 per cent glycerol, it retains the metachromatic property. The
metachromatic substance is therefore not easily washed out of muscle.  If, however,
a watery suspension of the washed muscle is heated to 65"C, then the juice, sepa-
rated from the particles, shows, on addition of toluidine blue, a metachromatic
color while the insoluble residue colors orthochromatically.  Hence, denaturation by
heating releases a metachromatic substance.  On adding the dye in sufficient quan-
tity to the filtrate, a metachromatic precipitate forms; the supernatant remains
blue indicating that the metachromatic material is precipitated quantitatively.
The precipitate can be separated by centrifugation and the dye dissociated from the
precipitated substance (see below).  The dye can thus be used for the simple isola-
tion of the metachromatic material.

This material is a protein.  It gives a strong biuret reaction, and is precipitated
by trichloracetic acid and ammonium sulfate (40 per cent of saturation).  It does
not precipitate on boiling but does so on addition of acetic acid to the hot solution.
It can also be precipitated, even at a high dilution, isoelectrically around a pH of
5.5-5.7 (addition of NaCl shifts it to a lower pH).  The isoelectric precipitation can
also be utilized for isolation purposes.  Molisch's test was negative.
That the protein is fibrous is indicated by its high viscosity and its double refrac-
tion of flow.  Addition of 0.1 M NaCl greatly decreases the viscosity.  Its aqueous
solution can be lyophilized.

The protein, which redissolves at higher ionic concentrations, is precipitated by
0.01 M CaC12 or MgC4.  At certain concentrations of NaCl or KCl, Ca has a
stronger precipitating action than Mg, a difference which deserves further study.
That the dye and the Ca or Mg precipi.tate one and the same protein, quantitatively,
is shown by the fact that the supernatant of the ionic precipitation gives no precipi-
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1034       HIOCHEMISTRY: SZENl'-GYdRGYI AND KAMINER   hoc. N. A. S.

tation with toluidine blue, and vice versa.  When a high concentration of mono-
valent or divalent ions is added to the metachromatic precipitate, it redissolves and
becomes orthochromatic.

That this protein may form part of the actomyosin system is suggested by the
following. Actomyosin (myosin B) in suspension stains metachromatically. If
the actomyosin is heated to 65OC!, it denatures and precipitates, whereupon the
metachromatic protein goes into solution, giving a metachromatic precipitate on
addition of toluidine blue.  The metachromatically stained actomyosin still super-
precipitates with ATP but at the same time becomes orthochromatic.  Actin (G or
F, prepared without special precautions for the elimination of tropomyosin) was
also found to be metachromatic. Myosin, on the other hand, did not give the
metachromatic reaction.

On the basis of its metachromatic property the protein described will be called
"metin," while its tertiary complex with actomyosin may be termed "metacto-
myosin."

Metin has several properties in common with tropomyosin4 and has also properties
common with Amberson's delta protein.5  I'urther studies are needed for its
characterization.

E&action Method.-Metin can be prepared from the muscles of a freshly killed
rabbit.  The skinned and eviscerated rabbit is briefly cooled on ice, its muscles
excised and minced on a precooled meat mincer.  The mince, say 500 gm, is sus-
pended in 1500 ml of OoC distilled water, gently shaken for several hr at O"C, and
then filtered through cheesecloth.  The extracted muscle is pressed out by hand,
suspended once more in 1500 ml of ice-cold water and, after shaking for several
hours, is again separatid using cheesecloth as before.  The muscle is then suspended
in 1000 ml of an aqueous 0.05 M NaCl solution, and rapidly heated to 65OC while
stirring vigorously.  The suspension is then cooled on ice, again passed through a
cheesecloth, and then centrifuged.  It usually gives a limpid supernate.  If no NaCl
is added, the filtrate is turbid.  If the muscle is washed insufficiently and the biva-
lent ions are not eliminated, the metin extracts poorly or not at all.

Metin can be precipitated from the extract in various ways.
Precipitation by toluidine blue:  The pH is adjusted to 7, and a 1 per cent watery
solution of toluidine blue is added6 till the supernate of a small sample is intensely
blue and gives no precipitate on addition of more dye.  This point is usually reached
with the addition of about 0.1 per cent dye.  The precipitate is separated by cen-
trifugation, washed with about 200 ml of water, and centrifuged again.  Then the
precipitate is dissolved in about 100 ml of a 20 per cent acetone containing 0.2-0.4
per cent acetic acid.  A homogenizer can be used with advantage. The dilute
acetone-acetic acid solution dissolves and dissociates the precipitate into protein
and the free blue dye.  The latter can be eliminated in various ways.  It can be
eliminated by adding powdered charcoal (Nor&A) under strong mixing. Since
the Norit does not bind the dye quantitatively, it is advantageous to add it only
until the fluid is discolored about halfway, then filter through a thin sheet of celite,
and repeat the charcoal treatment until practically all the dye is removed.  The
same can be achieved by dialyzing the solution against the same solvent with liberal
amounts of charcoal suspended in the outer fluid.

The metin can be precipitated from the discolored solution by adjusting the pH


VOL. 50, 1963   BIOCHEMISTRY: SZENFGYORGYI AND KAMINER        1035

to 5-5.7 and centrifuging out the isoelectrically precipitated protein, which readily
redissolves on neutralization.

Dr. Eloise E. Clark, to whom our thanks are due, examined such a preparation in
the ultracentrifuge and obtained a peak with a Gaussian distribution (S20,W = 2.85
X 10-l*; 4.7 mg/ml, 0.1 M NaCl).  On prolonged spinning, a minute quantity
of a slightly faster-sedimenting fraction (SZO,,,. = 3.6 X lo-la) was observed.
The greatest amount of the dye can also be eliminated by precipitating the protein
solution from the acetone-acetic acid by the addition of 4 volumes of acetone which
brings down the metin in the form of a voluminous precipitate.  The solvent is
decanted and the voluminous precipitate can be made to collapse by pressing it out
with a spatula.  The precipitated metin can now be slowly dissolved in neutral or
slightly alkaline water. Again, homogenization may be useful.  By adding butanol,
remnants of the dye may be shaken out of the watery solution.
Our preparation, at this stage, seems to contain traces of nucleotides present as
impurity, giving a weak absorption around 270 PM and a weak reaction for phos-
phate. (A pl t d d
       sam e s u ie contained 4.8 mg of protein and 1 pg of P per ml.)
Similar to other metachromatic substances, solutions of metin first color meta-
chromatically on the addition of small quantities of dye and then precipitate only
after the dye concentration approaches equimolarity with its binding sites.  Ap-
proximately one dye molecule is bound by ten amino acids of the metin.
Isoelectric precipitation:  Metin can also be precipitated from the primary muscle
extract isoelectrically.  The NaCl present shifts the isoelectric point to a lower pH.
Complete precipitation is obtained in presence of 0.05 2M NaCl at pH 4.5-5.  The
precipitate is separated by centrifugation and redissolved by neutralizing the sus-
pension. The protein thus prepared is precipitated quantitatively by toluidine
blue, indicating that it is identical with the protein obtained by the dye methods.
Precipitation by bivalent ions:  From a salt-free aqueous solution, the metin can
also be precipitated quantitatively by 0.01 M CaCh or MgC12.  This precipitation
is prevented by 0.05 M NaCI.  If the extraction of muscle was made in the absence
of NaCl, the Ca precipitate co-precipitates the substance which has caused the,
turbidity of the extract.  It can be separated from the latter by centrifugation after
redissolution. If the solution is still turbid, it can be clarified by adding 0.05 M NaCl
and 0.01 MgCh, and centrifuging, the salts being subsequently eliminated by iso-
electric precipitation.

+ This research was supported by a grant from the National Institutes of Health (GM-10383).
1 Heilbrunn, L. V., and F. J. Wiercinski, J. Cellular Camp. Physid., 29,15, (1947).
* Weber, A., and S. Wincur, J. Biol. Chem., 236,3198, (1961).
* By "metachromasia" is meant the property of macromolecules to be stained by certain dyes
with a color which is different from the orthochromatic color of the dye itself.  Toluidine blue, for
instance, at high dilution (10-e M), at which it is present as a monomer in water, has an absorption
at 623 mM, which corresponds to its blue orthochromatic color.  At a higher concentration (10")
it forms dimers which have a purplish tint and a somewhat weaker absorption at 586.  If, for
example, polyethylsulphonate of MW 12,900 gm (kindly supplied by Upjohn and Company, Kal-
amazoo, Michigan) is added, the absorption becomes considerably lower, wider, and shifts strongly
to a shorter wavelength (max 526)' as also is indicated by the metachromatic purple color.  There
is no acceptable general theory for this change.  Since dimerization shifts the absorption to a
somewhat shorter wavelength, it seemed reasonable to suppose that the greater metachromatic
shift is due to a further stacking of the dye molecules on the negative groups of the colloid.  This
theory was revived by F. Bradley, and M. K. Wolf in these P~OCEEDINW, 45, 844 (1953).  How-


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MATHEMATICS: HUEBSCH AND MORSE

PROC. N. A. S.

ever, it is easy to show that this theory is not generally acceptable.  If, for instance, a dilute
toluidine blue solution is added gradually to a relatively concentrated solution of a few per cent of
polyethylsulfonate, the Crst trace of dye added, detectable by the naked eye, is already meta-
chromatic. It seems unreasonable to suppose that this minute quantity of dye would form stacks
and not distribute itself among the great excess of SOS groups.  Similarly, if a solution of poly-
ethylsulfonate is gradually added to a solution of toluidine blue, the maximal metachromatic
shift is obtained when there is one SOa group for every dye molecule.  Thus, there being no
generally acceptable theory for metachromasia, one may sum up the situation by saying that while
the weak interaction between two dye molecules in a dimer causes a small shift and a small de-
crease in absorbancy, the stronger interaction between the basic dye and strongly negative SOr,
Pot, or COOH groups causes a greater shift with decrease and broadening of the absorption.
Though most proteins contain COOH groups they are not usually metachromatic, the number of
COOH groups being low and their electrostatic action being, possibly, also compensated by the
NH2 groups present.

4 Bailey, K., B&hem. J., 43,271, (1948).
6 White, J. I., H. B. Bensusan, S. Himmelfarb, B. E. Blankenhorn, and W. R. Amberson, Am.
J. PhpGoE., 188,212, (1957).

8 Other metachromatic dyes, lie acridine orange, can equally be used.