Ribonuclease amino acid composition

Fig. 2.—A Portion of the Proton-decoupled, Natural-abundance, 13C-N.m.r. Spectra of Model Compound 6 and Bovine Ribonuclease B at 67.9 MHz. [(A) Compound 8 in HzO (25 mM, pH 6.5) after 8192 scans (2-s recycle-time) (B) spectrum of ribonuclease B after digital subtraction of the spectrum of ribonuclease A. (This enzyme has the same amino acid composition as ribonuclease B, but contains no carbohydrate.) Spectra were taken from Ref. 27.1...

CHW Hirs, WH Stein, S Moore. The amino acid composition of ribonuclease. J Biol Chem 211 941-949, 1954. 

The total number of theoretically exchangeable hydrogens, H, can be computed from the amino acid composition of the protein. An illustrative calculation is shown in Table XXII for ribonuclease at low pH, say pH 1, where it contains its maximum number of protons. The amino acid composition is that of Hirs et al. (1960). The total number of backbone, side-chain, and end-group hydrogens is 256 at pH 1.0. If the exchange experiment is carried out at any other pH, then a correction must be applied,... 

The term molecular weight denotes the relative molecular mass (M,), which is the ratio of the mass of a molecule to one-twelfth of the mass of carbon 12. The Mr is dimensionless for example, the M, of ribonuclease A is 13,680. The Mr of a protein can be calculated as the sum of the molecular weight of its constituent amino acids when the primary sequence or the minimal amino acid composition is known. 

Hydrolysis and subsequent amino acid analysis can be used to determine the composition of a protein as well as quantify concentration. Essential to the methodology is the initial hydrolysis step during which the peptide bonds are broken under acidic conditions at high temperatures to generate free amino acids. Conventional protein hydrolysis is typically performed with 6 N HCl at 110 °C for 24 h. The first published study on the use of microwave heating for acid hydrolysis of proteins appeared in 1987, ribonuclease A and insulin B being used as substrates. Solutions were heated in a domestic microwave oven for periods of 1 to 7 min. Amino acid recoveries after heating for 3,4, and 5 min corresponded... 

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