Determination of myofibrillar and connective tissue protein contents of young and adult avian (Gallus domesticus) skeletal muscles and the N tau-methylhistidine content of avian actins.

The myosin, actin, and collagen contents of young and adult avian red (leg) and white (breast) skeletal muscles from White Leghorn chickens have been determined by the use of analytical chromatographic methods developed to quantify the unique amino acids that occur in these proteins. Because one mole of actin purified from the red and white muscles of Leghorn chickens and one mole of myosin contain respectively one and two moles of N tau-methylhistidine, and the molar ratio of myosin and actin in skeletal muscle is known to be 1:6, the myofibrillar myosin and actin contents of avian skeletal muscles can be determined from the amounts of protein-bound N tau-methylhistidine found in acid hydrolysates of this tissue. Actin accounts for an estimated 11.2 to 12.2% of total muscle mass in both muscles or about 21.1% of total myofibrillar protein, whereas myosin ranges from 23.4 to 25.8% of total protein, corresponding to a mean value of 43.8% of total myofibrillar proteins. Total avian collagen ranged from 1.96 to 3.08% in breast and from 5.63 to 6.87% in leg skeletal muscles. With the methods described herein, content of collagen and collagen-like proteins can be calculated from amounts of 5-hydroxylysine present. The content of total connective tissue proteins could also be calculated from amounts of 4-hydroxyproline found. These quantifications are based on total protein content of the selected avian muscles determined by their detailed amino acid composition.

Determination of methylated basic, 5-hydroxylysine, elastin crosslinks, other amino acids, and the amino sugars in proteins and tissues.

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.

Rapid method for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine and the amino sugars in proteins and tissues.

A rapid and sensitive chromatographic method is described for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine, glucosamine and galactosamine at picomole levels in protein and tissue hydrolysates. This method uses either an automated amino acid analyser with a 17.5 X 0.28 cm microcolumn packed with 6.0 +/- 0.5 micron spherical resin, thermostated at 52 degrees C, one buffer system (0.21 M sodium citrate, pH 5.125) and 3-nitrotyrosine as the internal standard, or conventional instruments using the same system but with larger diameter columns and resins (11.0 +/- 1.0 micron). This method should be especially valuable for determining collagen and elastin in tissue hydrolysates from the amounts of 5-hydroxylysine, and desmosine or isodesmosine present, respectively, and for studying protein hydroxylation, glycosylation, cross-linking formation, and the turnover rates of collagen and elastin in normal and diseased tissues.