ISOLATION AND CHARACTERIZATION OF TITIN T1 FROM BOVINE CARDIAC-MUSCLE

Methods were developed for isolation of bovine cardiac titin in the ap parently full-sized T1 form. The pure protein could be obtained either in the denatured state after chromatography of myofibril extracts on Sephacryl S-1000 or in the native state using hydroxylapatite chromato graphy. Titin-specific chicken polyclonal antibodies against denatured titin stained the I band regions and weakly stained the M line area o f cardiac myofibrils but primarily stained the A-I junction positions in skeletal muscle myofibrils. Bovine cardiac titin contained 176 +/- 5 mu mol of sulfhydryl groups per gram in the presence of 6 M guanidin e hydrochloride or O.1% SDS; only 45% of these groups were reactive un der non-denaturing condition. Circular dichroism measurements indicate d that titin contained approximately 53% beta-sheet and 47% random coi l at pH 7.0. These results are consistent with the idea that titin is primarily composed of immunoglobulin C2 and fibronectin type III like motifs throughout its length, including the flexible I band region.