Binding of ions by myosin. I. Note on the binding of methyl orange by myosin

Binding

of Ions by Myosin. I. Note on the Binding Methyl Orange by Myosinl E. Mihfilyi

From the Institute

of

and B. N. Ghosh

for Muscle Research, at the Marine Hole, Massachusetts Received

Biological

Laboratory,

Woods

May 26, 1952

I~R~DUOTI~N The interaction of methyl orange with proteins has been widely investigated by Klotz and his associates (1,2,3). Under identical experimental conditions various proteins showed very different affinities toward the dye anion,.thus permitting their arrangement in a specific sequence according to their binding abilities. The binding of methyl orange was chosen as a measure of the interaction between anions and proteins, because it is intermediate between the very strong binding of some anions, e.g., dodecyl sulfate, and the very slight binding of others, e.g., chloride, neither of the extreme situations being favorable for the detection of relative differences in the binding capacity. Klotz (3, 4) attempted to correlate the affinities toward anions with the amino acid composition of the proteins, and was able to calculate from the latter a “binding index” which was successfully compared with the experimentally established binding abilities. As an introductory work to the study of the interactions between myosin and ions it seemed desirable to investigate the binding of methyl orange to myosin, to establish the position of this protein among those investigated by Klotz et al., and to compare at the same time the experimental result with the calculated binding index. EXPERIMENTAL Myosin was prepared in the crystalline form according to SzentGyiirgyi (5). The equilibrium dialysis experiments were performed as closely as possible to the standard conditions described by Klotz and Urquhart (3). The slight solubility of myosin at low ionic strengths 1 Sponsored by a grant Heart Association.

from Armour

& Co., Chicago, 104

Ill.;

and The American

BINDING

OF

IONS

BY

MYOSIN’.

I

105

made it impossible to perform the determinations on dissolved myosin at 0.1 ionic strength, therefore the higher ionic strength of 0.3 was used. In some experiments 0.04 M potassium phosphate buffer of pH 6.8 was present; in others there was no buffer, the pH of the myosin solution being adjusted to 6.8 and checked before and after the dialysiswith a

1.6 -

0.8 -

6 -log

5

free methyl orange

4 cont.

FIG. 1. Binding of methyl orange by myosin (curve 3). For comparison the binding curves of bovine serum albumin (curve 1) and lactoglobulin (curve 2) taken from the paper of Klotz et al. are also shown. 0rdinste:‘moles of methyl orange bound per lo6 g. protein. Abscissa: logarithm of free methyl orange concentration.

glass electrode. To increase the accuracy of the results, in the second series of determinations the protein concentration was increased from 0.2 to 1.0%. The dialysis in cellophane bags was continued for 72 hr. at 0°C. In Fig. 1 the number of bound methyl orange molecules has been plotted against.the logarithm of the free dye concentration. All the experimental points fall on the same curve, showing that neither the pres-

106

E. MIHtiLYI

AND B. N. GHOSH

ence of phosphate, nor the increase of the protein concentration affected the extent of the binding. The figure also shows the curves obtained by Klotz and Urquhart (3) with bovine serum albumin and &lactoglobulin. With all three proteins the results are expressed on a common basis, as moles of methyl orange bound/l05 g. prot,ein. As can be seen, myosin has Its affinity a lower affinity for the dye anion than does /3-lactoglobulin. is higher than that of egg albumin and a number of other proteins which under the above conditions do not bind any methyl orange. The binding index was defined by Klotz as 2/N=/ Z;(COO-1 - BJOH-1 Since its calculation involves the difference of two fairly large numbers situated rather close together, the figures used should be known very accurately. This is certainly not the case with myosin. Bailey’s (6) analytical values give figures somewhat lower than those obtained by titration. He gives 139 total cationic, 103 hydroxyl, and 131 free carboxylic groups per lo6 g. myosin. The isoelectric point of myosin shows clearly that the number of free COO- groups should be somewhat greater than that of the total cationic groups. Titration methods gave 150 total cationic (7) and 165 free COO- groups (8). The binding index calculated with the analytical figures is 4.8. With the titration data it, becomes much smaller, 2.4, corresponding to the slight binding of methyl orange found experimentally. Myosin, therefore, seems to fit into the table given by Klotz and Urquhart, where lactoglobulin has.a binding index of 4.6 and egg albumin of 2.5. This is remarkable because the structures of all of the proteins investigated by the above authors are more or less symmetrical, in sharp contrast with the highly asymmetrical structure for myosin. REFERENCES 1. KLOTZ, I. M., WALKER, F. M., AND PIVAN, R. B., J. Am. Chem. Sot. 33, 1486 (1946). 2. KLOTZ, I. M., AND URQUHART, J. M., J. Am. Chem. Sot. 71, 847 (1949). 3. KLOTZ, I. M., ANY URQUHART, J. M., J. Am. Chem. Sot. 71, 1597 (1949). 4. KLOTZ, I. M., Cold Spring Harbor Symposia Quant. Biol. 24, 97 (1950). 5. SZENT-GY~RGYI, A., Chemistry of Muscular Contraction, 2nd Ed., p. 146.

Academic Press, New York, 1951. 6. BAILEY, K., Biochem. J. 43, 271 (1948). 7.. EDSALL, J. T., GALE, A., AND PERLMANN, G., quotedinCoHN, E. J., AND EDSALL, J. T., Proteins, Amino Acidsand Peptides, p. 355. Reinhold Publishing Corp., New York, 1943. 8. MIH~LYI, E., Enzymologia 14, 224 (1950).