X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method

Area of Science:

• Muscle physiology
• Biophysics
• Biochemistry

Background:

• Preserving native muscle structure is crucial for understanding filament organization.
• Chemical skinning methods are explored to obtain high-quality X-ray diffraction patterns.

Purpose of the Study:

• To evaluate Triton X-100 for chemically skinning frog sartorius muscles.
• To assess the retention of native muscle structure after skinning.
• To investigate myofibril swelling and its reversal.

Main Methods:

• Chemical skinning of whole frog sartorius muscles using Triton X-100.
• X-ray diffraction analysis of skinned muscle fibers.
• Measurement of myofibril swelling and swelling pressure using colloids (PVP, dextran).

Main Results:

• Triton X-100 skinning (2h) largely retains native fibril and filament structure for X-ray diffraction.
• Myofibrils swell after detergent skinning, a phenomenon reversible with high molecular weight colloids.
• Swelling pressure was estimated at 35 Torr in KCl-based relaxing solution; acetate reduced swelling compared to chloride.
• Loss of constant-volume relation between sarcomere length and lattice spacing observed.
• Intensity changes in X-ray patterns (I1,0 rise, I1,1 fall) were not calcium-dependent, suggesting thin filament disordering.

Conclusions:

• Chemical skinning with Triton X-100 is a viable method for preserving muscle structure for X-ray studies.
• Myofibril swelling is an inherent property of detergent-skinned muscle, manageable with osmotic support.
• Observed X-ray intensity changes are attributed to thin filament disordering, not cross-bridge activity.