Heterogeneity of human myoglobin

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Heterogeneity

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of Human Myoglobinl

Observation on the inhomogeneity of crystallized myoglobin (Mb) of some animals (whale, ox, horse) exist in literature (1, 2), but it is only very recently that Theorell and Akeson (3) succeeded in showing that crystallized horse Mb contains three different fractions which can be separated only by means of electrophoresis. Similar results were obtained later by Boardman and Adair (4) using ion exchange chromatography on Amberlite resin. Human Mb had still not been examined from this point of view, moreover the existence of different myoglobins had not yet been directly demonstrated in crude muscle extract. In fact all the results of the research carried out so far on different myoglobins refer to preparations of pigment obtained after crystallization or fractional precipitation with ammonium sulfate. In this paper we report the results of research carried out with the aim of ex-

FIG. 1. Eleetrophoresis of human muscle extract (A), and of crystallized man Mb (B). The Mb solutions were treated with KaFe(CN)B , to transform the pigment in the ferric form. Other conditions as in the text.

huall

amining the homogeneity of human myoglobin, both on crystallized preparations and directly on muscle extract, employing an opportunely developed electrophoretic technique which has also been successfully used for the determination of small quantities of myoglobin and hemoglobin (5). As a supporting medium for the electrophoresis we used Whatman no. 1 paper previously soaked in a solution of 0.5’% egg albumin and dried in a current of warm air; in this way a film of denatured albumin forms on the paper, preventing adsorption phenomena. The electrophoresis was run in a Verona1 buffer pH 8.6, ionic strength 0.01-0.03 with 309 volts/Z1 cm. for a period of 6-8 hr. Under these conditions hemoglobin remains almost on the start line while myoglobin moves (by electro-osmosis) towards the cathode. After the electrophoresis run, the paper, which is dried in a current of warm air, is sprayed with a benzidine reagent (alcohol 9570, 5 ml.; benzidine 0.1% in methanol, 1 ml.; HsOz 3OoJ 1 ml.; glacial acetic acid, 0.3 ml.). 1 Aided by a grant

from the Rockefeller

Foundation.

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FIG. 2. 1 = Mb II; 2 = unhomogenous

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crystallized

human Mb; 3 = Mb I

Thus we succeeded in investigating the electrophoretic behavior of the myoglobin even with the smallest quantities of pigment (5-10 pg.) both in pure solutions and also in muscle extracts. By employing this technique we were able to show the presence of three different EL El

; 1,Oo-Mb n- Mb1 o-Mbn

0, 5-

OL 480

500

520

540

560

580

600

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640 Amp

FIG. 3. Absorption curves of Mb I and Mb II, after elution from electrophoresis, and of crystallized human Mb as ferric compounds (Mb).

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EA ER max l,( I-

O,!5-

o-Mb A-Mb1 o-Mbn

!I-

1

0

500

520

540

I

I

I

560

580

600 nm)l

FIG.

4. Absorption curves of Mb I, Mb II, and crystallized human Mb as CO compounds.

components (Mb I, Mb II, Mb III) both in solutions of crystallized human myoglobin and in water extracts of human muscle (from which the hemoglobin had, for the most part, been removed by means of washing with water). An example of these experiments is reported in Fig. 1. Densitometric examination of the spots showed that the relative percentages of the three components are as follows: for the crystallized myoglobin Mb I 740/ Mb II 19’%, Mb III 7%, for the muscle extract Mb 1830/ Mb II 12%, Mb III 5%. Mb I and Mb II have been isolated by the preparative electrophoresis, on Mtinktell no. 20 paper, of highly concentrated (23%) solutions of crystallized adult human Mb and the consecutive elution of the single components with water.

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Figure 2 shows that Mb I and Mb II are eleetrophoretically homogeneous and that they have an electrophoretic behavior identical to the components not isolated. Moreover spectrophotometric analysis of the solutions of Mb I and Mb II shows that the two components have absorption curves almost identical to each other and which correspond to those obtained from crystallized human myoglobin (Figs. 3 and 4). Research carried out with the same technique have revealed the presence of three different myoglobins also in the muscle and in the crystallized myoglobin of fish (Thunnus thynnus, Palamys sarda). Detailed research will be published in a work in extenso. The authors wish to thank Miss S. Benerecetti for skilled technical assistance. REFERENCES 1. 2. 3. 4. 5.

SCHMID, K., Nature 163, 481 (1949). LEWIS, U. J., AND SCHWEIGERT, B. S., J. Biol. Chem. 214, 647 (1955). THEORELL, H., AKESON, A., Ann. Acad. Sci. Fennicae 60, 303 (1955). BOARDMAN, N. K., AND ADAIR, G. S., Nature 177, 1078 (1956). MONDOV~, B., ANTONINI, E., AND ROSSI-FANELLI, A., Intern. Congr. Biochem. Abstr. of Commun.; 3rd Congr. Brussels, 1966, p. 81.

Chemistry, In&it&e of Biological Cniversity of Rome, Rome, Italy Received September 21, 1966

A. ROSSI-FANELLI E. ANTONINI