A-band mass exceeds mass of its filament components by 30-45%

We have measured filament lattice spacing in fibrils using X-ray diffraction, and find that STEM-determined mass/length values reported for myofilaments should give 13% w/v as the filament protein concentration in the lattice of the AO-band (filament overlap zone) of both insect flight muscle (IFM) and vertebrate skeletal muscle ( VSkM ). This is well below the actual mass concentration of AO-bands as measured by immersion refractometry of detergent-washed rigor myofibrils under the phase microscope. This technique identifies an immersion fluid of suitable refractive index (RI) for matching out all image contrast between background and the selected cross-band. We used RI fluids in which the effective RI matching component is a large-particle solute (Percoll or hemocyanin) excluded by the filament lattice. The measured RI indicates that protein concentration in AO-bands is 16-17% in Lethocerus and other IFM fibrils including CAF-digested fibrils, and is 18-19% in rabbit VSkM fibrils. On IFM fibrils we also measured absolute buoyant density in Percoll as greater than or equal to 1.042; this supports the value for mass concentration as determined by RI. The mass discrepancy between fibrils and filaments does not seem to arise from faults in the methods used. We therefore accept the STEM-determined mass/length values for thick filaments which indicate 4 myosins/crown in IFM and 3 in VSkM , and we believe there is considerable extra nonfilament material (concentration: 30-50 mg/ml) between the filaments in fibrils. In stretched VSkM , it is the H-bands, not the I-bands, which have an excess over filament mass content. The extra mass has not been identified.