Quantitative N-terminal Analysis Research Articles

A complete quantitative N-terminal analysis method

A complete quantitative N-terminal analysis (QNA) technique based on the application of dimethylaminoazobenzene isothiocyanate is described. The method allows recovery of all free N-terminal amino acids, including Asn, Gln, Trp, Ser, and Thr in quantitative yield. N-Termini of polypeptides as little as 5 pmol can be reliably and reproducibly determined by this method. This QNA method is useful in many aspects of protein structure analysis. (a) QNA is useful in assessing the purity, identity, and quantity of a polypeptide preparation. It has also been applied in our lab as a routine guarding step to prevent impure or ill-characterized samples from occupying the space of the gas-phase sequenator. (b) QNA of a trypsinized protein generates a miniaturized amino acid composition which is useful both in characterizing the identity of a protein and in comparing the homology of structurally related proteins. (c) QNA can be used to follow the pathway and preferential cleavage sites of limited proteolysis. (d) QNA is useful in characterizing selectively modified Lys and Arg residues. The details of this QNA method and the results of its applications are presented here.

The active form of the cytochrome d terminal oxidase complex of Escherichia coli is a heterodimer containing one copy of each of the two subunits.

The cytochrome d complex is a component of the aerobic respiratory system of Escherichia coli. The enzyme functions as a terminal oxidase, oxidizing ubiquinol-8 within the cytoplasmic membrane and reducing oxygen to water. The enzyme is of particular interest because it is a coupling site in the electron transfer chain. The electron transfer reaction catalyzed by this enzyme is coupled to the translocations of protons across the membrane (H+/e-approximately equal to 1). The oxidase contains two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, with molecular weights of 58,000 and 43,000. In this paper, the question of the quaternary structure is addressed. Quantitative N-terminal analysis of the isolated enzyme and relative mass quantitation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the subunits are present in equimolar amounts. Sedimentation velocity and sedimentation equilibrium studies were used to characterize the hydrodynamic properties of the purified enzyme solubilized in Triton X-100, under conditions where the enzyme is active. It is concluded that the active enzyme in Triton X-100 is a heterodimer, containing one copy of each subunit. This is likely the structure of the enzyme in the E. coli membrane.

Chemical modification of the carboxyl groups of protein substrates enhances their thrombin susceptibility

Native or denatured protein substrates which are hardly digested by thrombin can become much more efficiently cleaved by the enzyme after chemical modification of their carboxyl groups. Five antibody κ-chains were used to demonstrate this effect. The selective cleavage sites were determined by quantitative N-terminal analysis and N-terminal sequencing. All five κ-chains share the same cleavage sites at Arg-Thr (residues 108–109), Arg-Glu (residues 142–143, Glu side chain modified with glycine amide), Lys-Ser (residues 207–208) and Arg-Gly (residues 211–212). One of the major cleavage sites (Arg-Thr) is located at the joint of the variable/constant region. The amino acids adjacent to these cleavage sites underline the proposed structural requirements for a potential thrombin substrate [(1985) Eur. J. Biochem. 151, 217–224]. This approach can facilitate the application of thrombin in generating large polypeptide fragments of proteins.

Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N-terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.

An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart.

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.

Fibrinaemia and multiple thrombi in pancreatic carcinoma. A case studied with quantitative N-terminal analysis.

In a 64‐year‐old man several thrombo‐embolic episodes occurred in spite of oral anticoagulant treatment with Thrombotest values below 10%. Normalization of a low plasma fibrinogen, cryofibrinogen and the ethanol gelation test during heparin treatment, suggested chronic intravascular coagulation several weeks prior to the recognition of a pancreatic carcinoma. N‐terminal analysis of the clottable plasma proteins established that about 11 % of the plasma fibrinogen existed as soluble fibrin in plasma. Such a large amount of soluble fibrin suggests the joint action of thrombin and other proteases on the clottable proteins in vivo.