Disulfide Pairs Research Articles

A novel concatenated dimer of recombinant bovine somatotropin.

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.

Complete assignment of neurophysin disulfides indicates pairing in two separate domains.

The pairing of the 14 half-cystine residues of bovine neurophysin was established by sequential proteolytic digestion. Purified released peptides and the residual disulfide-linked core were monitored at each step by use of amino acid analysis, gas-phase sequencing, and mass spectrometry. The approach included application of gas-phase sequencing to assign disulfide pairs in peptides containing multiple disulfides. The results demonstrate that neurophysin disulfides are paired in two distinct domains--an NH2 domain (residues 10-54) containing four disulfides and a COOH domain (residues 61-85) containing three disulfides. The specific disulfide bridges are Cys-10 to Cys-54, Cys-13 to Cys-27, Cys-21 to Cys-44, Cys-28 to Cys-34, Cys-61 to Cys-73, Cys-74 to Cys-79, and Cys-67 to Cys-85. The results place the internally duplicated segments of neurophysin (residues 12-31 and 60-77) in separate domains. Disulfide-pairing patterns within each domain are homologous with the exception of the Cys-10 to Cys-54 bond, which is unique to the NH2 domain and which links the two ends of this domain together. The potential role of the Cys-10 to Cys-54 bond in organizing the hormone-binding site is discussed.

Application of high-performance liquid chromatography in neurophysin disulfide assignment

The combined use of ion-exchange, and reversed-phase high-performance liquid chromatography (HPLC) for the isolation of cystine-containing peptides from highly heterogeneous products of the proteolytic digestion of bovine neurophysins is described. The protein was sequentially cleaved by enzymes of decreasing specificity; the peptides released were initially fractionated by gel chromatography and then purified by HPLC. The purified peptides were analyzed by determination of their amino acid composition and mass spectrometry, supported by sequencing techniques. Three of the seven disulfide pairs of neurophysin have now been assigned. The usefulness of the combined use of HPLC and mass spectrometry in assigning these and the other disulfide pairs is illustrated.

Partial assignment of disulfide pairs in neurophysins

The original report assigning the pairing of neurophysin's 14 half-cystine residues (Schlesinger et al. (1972), Proc. Natl. Acad. Sci., U.S.A., 69 ,3350–3353) was based on an incorrect amino acid sequence. In the present study, re-investigation of the results of proteolytic fragmentation of bovine neurophysins indicates that the majority of the original assignments were incorrect. Three disulfide pairs are now assigned as Cys 21-Cys 44, Cys 67-Cys 85 and Cys 74-Cys 79. The pairing pattern indicates that neurophysin's variable carboxyl terminal region, separately encoded by the third gene exon, does not form a self-contained domain.

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion.

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.

A convenient assay method for disulfide interchange and other thiol transfer reactions

A convenient method is presented for quantitating disulfide interchange reactions based on the use of a commercially available organomercurial resin. Isotopically labeled thiol is incubated with a disulfide and the reaction is terminated by charging the incubation mixture onto the resin. The thiol is bound as the mercaptide allowing labeled interchange products, as well as the unlabeled interchanges substrate, to be eluted for quantitation. In principle the method is applicable to all monothiol and disulfide pairs and may be more generally applied to other thiol-transfer reactions that result in conjugation of the thiol. Examples of the use of the technique for glutathione transferase-catalyzed reactions are presented.