X-RAY DIFFRACTION STUDIES ON FROG MUSCLES
Authors: Spiegel-Adolf M. , Henny G. C. , Ashkenaz E. W. 
Published: May 2004

1. X-ray diffraction studies of sartorius muscles of Rana pipiens were made in a new x-ray diffraction camera which permits exposures of 3 to 6 minutes. The object-film distance can be varied from 20 to 80 mm; the muscle inside the camera can be electrically stimulated while contracting isotonically or isometrically, and can be observed by a special device. After exposures up to 30 minutes (approximately 40,830 r) muscles are still alive and responsive.
2. Contrary to the x-ray diffraction pattern of powdered dry muscle, which pattern consists of two rings corresponding to spacings of 4.46 Å.u. and 9.66 Å.u., both moist and dried whole sartorius muscle show signs of orientation in both rings, consisting of two equatorial streaks (wet) or points (dry) and meridional sickles. The moist muscle shows in addition a diffuse water ring. The spacings corresponding to the orientation points and elliptical structure show only slight differences in moist and dried samples. Through statistical computations based on two different series consisting of thirteen moist and twenty-eight dried samples, and nine muscles before and after drying, it was shown that only the divergence in the smaller spacing has some real significance, which indicates that most water of the moist muscle is bound intermolecularly. Upon resoaking of dried muscle the x-ray diffraction pattern of the moist muscle is restored.
3. Stretching of muscle by weights below the breaking point produces an additional well defined diffraction line, corresponding to a spacing of 4.32 Å.u. A similar diffraction line can be produced in frog tendon upon stretching.
4. The influence of heat on the x-ray diffraction pattern of muscle depends upon the maximum temperature and the length of action; 5 minutes at 50° C. markedly reduces the orientation of the sample; 5 minutes' immersion in boiling Ringer's solution destroys the orientation and produces a ring corresponding to a spacing of 5.3 to 5.5 Å.u. in the moist and sharpening of the backbone reflection in the dried specimen.
5. Ultraviolet light brings forth changes in the x-ray diffraction pattern varying with the intensity of the irradiation. Ultimately a disappearance of the equatorial points and of the outside sickles is achieved while the elliptical shape of the outside ring and its diffuseness persist. In addition two salt rings characteristic of NaCl indicate that the irradiated muscles have become permeable to the surrounding medium (Ringer's solution).
6. Both faradic and single shock electrical stimulation were tried on muscles. If shortening of the muscle is prevented either by sufficient weight or by tying the muscle in a frame, no changes in the x-ray diffraction pattern occur; if the muscle is allowed to shorten without weights or by using insufficient weights, then the orientation either disappears completely or partially. When the muscle is stretched while contracted by electrical stimulation the orientation of the x-ray diffraction pattern reappears.
7. A number of salts with uni- and bivalent ions in concentrations corresponding osmotically to 0.73 per cent NaCl and 10 per cent NH4Cl were studied in their effects upon the x-ray diffraction of muscles. Of the salts with univalent ions in the lower concentration only KCl causes a marked decrease of orientation and an increase in the permeability of the fiber membranes. Similar effects on the orientation seem to be produced by CaCl2 while MgCl2 causes rather a more pronounced orientation. At hypertonic salt concentrations the orientation disappears completely and the corresponding salt rings become visible. Besides, NaCNS seems to have a specific effect on the outside ring and LiCl produces a ring at 21.3 Å.u. and a splitting of the outside ring.
8. Strong mineral and lactic acids in concentrations up to 0.005 N have little if any influence upon the x-ray diffraction of muscles. A further increase in acidity to 0.01 N and above destroys the orientation completely, causes sharpening of the backbone reflection, and increased membrane permeability. These changes are irreversible upon neutralization. Also the effects of swelling upon the water ring of fresh muscle become manifest. Weak acids at higher concentrations show an effect similar to that of strong acids.
9. Rigor mortis produces a more or less complete loss of orientation. The muscles show signs of increased permeability.
10. Alkalies destroy the orientation of the x-ray diffraction pattern. The effective concentration is higher than the corresponding amount of acid.
11. Formaldehyde produces only minor changes in the x-ray diffraction patterns of muscles.
12. The effects of alcohol depend primarily upon the concentration applied. Low concentrations (5 per cent) seem to have a passing stimulating effect, at concentrations of 15 per cent, the anesthetizing effect becomes manifest in well defined orientation. The diameter of the water ring is reduced. If 95 per cent alcohol is allowed to act upon muscle for more than 12 minutes, then the orientation disappears completely and the backbone spacing becomes as sharp as in boiled muscle.
13. The effects of chloroform depend upon whether the muscle is allowed to contract or not. Only if the muscle is allowed to contract in chloroform-saturated Ringer's solution is the orientation lost and salt rings appear as well as a ring corresponding to a spacing of 22 Å.u,, which has been observed in other changes in muscles.
14. In muscles allowed to shorten in a caffeine-Ringer's solution the orientation disappears, salt rings become visible as well as a decrease in size of the water ring; a new arc corresponding to a spacing of 4.18 Å.u. was observed in one case.