摘要:

The major lethal component of the venom ofNaja naja philippinensishas been extracted and extensively purified, up to complete homogeneity, by physical and chemical standards and by exacting immunological tests. The method of preparation, aimed at preserving the native conformation, avoids the polystyrene resins commonly used for such toxins, but relies exclusively on hydrophilic supporting media and takes advantage of the extreme affinity of salt‐free neurotoxin for wetted poly‐dextran gels in order to increase the efficiency considerably. The pure toxin has an LD50of 0.05 μg/g, is a single chain polypeptide of 61 residues, has a mol. wt. of 6867, a pI of 10.8, and an unusually high absorptivity, 14,300 at 280 nm, when compared to all known parent toxins. At 10‐4to 10‐5M and slightly acidic pH, equilibrium sedimentation failed to provide evidence for dimerization or conformational heterogeneity. This minute and tightly assembled protein molecule belongs to type I neurotoxins of snake venoms, acting selectively by inhibiting myofibrillar membrane depolarization in excitatory synapses and blocking myoneural transmission to striated muscles, particularly those involved in pulmonary ventilation. This neurotoxin brings about two new and unexpected features: 1)It differs from all known isotoxins of type 1 by having Ala in its composition, located at position 11 in the sequence, an amino acid so far considered as typical of type II neurotoxins of more recent origin, and of all other isotoxins of higher mol. wt. 2)Strikingly, it contains two Trp instead of the unique Trp characteristic of all neurotoxins described so far. Though the unique ancestral Trp has been claimed repeatedly to play a critical role in lethality of all neurotoxins, the present sidewise duplication shows no influence on the LD50.ORD‐CD profiles display β sheet structure predominantly, an observation consistent with the occurrence of a majority of non helix‐formers in the two largest stretches. A third large loop, where hydrophobic and all the aromatic side chains are packed, may have helical conformation. Though denaturation in 6 M guanidine‐HCl releases completely randomized structures, as apparent by ORD‐CD, the unfolding in 10 M urea is far from complete and remains fully reversible. Fluorescence investigations by reversible solvent perturbation using methanol or urea confirm localized unfolding accompanied by the translation of the unique Tyr chromophore from the interior out to the surface of the molecule, whereas both Trp chromophores appear unaffected except for minor hyperchromatism. These residues may not necessarily be directly involved in the receptor‐complex formation, but may be essential for conformational stabilization. Whether neurotoxin engages in this binding process as a rigid ligand or by some kind of “induced‐fit” is a matter f

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1974.tb02380.x

出版商:Blackwell Publishing Ltd    

年代:1974

数据来源: WILEY

摘要:

A procedure, recently developed in this laboratory, for the rapid determination of the positions of disulfide bonds in proteins of known amino acid sequence was applied to the assignment of the disulfide bonds of ovine lactogenic hormone and human growth hormone. Each protein was hydrolysed by pepsin under conditions where disulfide interchange does not occur. Peptide maps of the resulting hydrolysates were prepared. Sodium borohydride reduction and subsequent treatment with S,S'‐dithiobis(2‐nitrobenzoic acid) (DNTB)revealed the locations of cystine‐containing peptides: four for ovine lactogenic hormone and three for human growth hormone. These cystine‐containing peptides were eluted, hydrolysed, and their amino acid compositions determined. By comparison of the analytical data with the known amino acid sequences around each half‐cystine residue and the known substrate specificity of pepsin, the positions of the disulfide bridges were unequivocally determined. The results confirm the disulfide linkages previously established by other methods. The scope and limitations of this procedure are

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1974.tb02381.x

出版商:Blackwell Publishing Ltd    

年代:1974

数据来源: WILEY

摘要:

A Fortran computer program to aid in disulfide bond analysis in proteins has been developed. The program evaluates amino acid analysis data from isolated cystine‐containing peptides by comparing it with known sequences around pairs of half‐cystine residues in the primary structure of a protein and recording all matches. The method involves presetting upper and lower bounds for each amino acid present in a cystine‐containing peptide by considering both partial degradation during acid hydrolysis and contamination sources for certain amino acids. Initially, limiting path terminals for each half‐cystine residue in the primary structure are determined by examining the surrounding sequences in both directions for “alien” amino acid residues, not present in the composition of a given cystine‐containing peptide. Subsequently, pairwise examination proceeds through several cycles incorporating minimal path and upper bound routines with intermittent revision of path ends. Finally, all pairs of half‐cystines whose surrounding sequences fall within the preset bounds for this peptide are listed. If too many possible pairs or none are found, the bounds are revised. The lists for all available cystine‐containing peptides are graphed to facilitate final determination of disulfide bond positions in the native protein.The computer program provided finite disulfide bond assignments for ovine lactogenic hormone and human growth hormone. In the case of hen egg‐white lysozyme, a considerable reduction was obtained from the large number of theoretically possible disulfide bond combinations to a few alternatives, and one of the four disulfide bonds was unequivocally determined. To assign the positions of the other three disulfide bridges, additional considerations, not included in the program, were applied, such as specificity of the enzymes used in digesting the protein and electrophoretic mobility of the ensuing cystine

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1974.tb02382.x

出版商:Blackwell Publishing Ltd    

年代:1974

数据来源: WILEY

摘要:

The influence of pH, temperature and the concentrations of zinc ions and reduced proinsulin upon the yields of reoxidized proinsulin were studied using the insulin radioimmunoassay. The optimal conditions were found to be pH 8.5, absence of zinc ions and a low protein concentration. However, even at a concentration of reduced proinsulin of 1.47 μM, the reoxidized proinsulin did not compete with125I‐insulin for the insulin antibodies as strongly as did genuine proinsulin. At high concentrations of reduced proinsulin(14.7 μM),the yield increased with decreasing temperature; at a 10‐times lower concentration the temperature had no influence upon the yields. Adsorption of reduced prosinulin to glassware was either irreversible or had a denaturing effect rendering the products immunologically inactive. Spontaneous reoxidation of reduced proinsulin is a rather delicate process, and the optimal conditions are different from those presentin vivo.When the reduction of proinsulin was carried out at a concentration of 31.5 μM, zinc ions were found to stabilize the reduced protein against denaturation and precipitation for more than 24 hours. However, at this concentration, reduced proinsulin is in a thermodynamic ally unstable state, both with and withou

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1974.tb02383.x

出版商:Blackwell Publishing Ltd    

年代:1974

数据来源: WILEY

摘要:

Solutions of arachin, a groundnut protein, show liquid‐liquid phase separations in certain conditions of ionic strength and pH. Phase diagrams show upper, lower and closed boundaries corresponding to salting in and salting out curves of globulins and a hitherto undescribed solubility behaviour. The protein rich liquid phases and protein ‘precipitates’ are similar.As conditions approach the phase boundary, arachin undergoes self‐association, as revealed by the concentration dependence of the sedimentation coefficient and gel chromatography, while in D2O, association is more extensive and the phase boundaries are correspondingly shifted. Oligomer formation could lead to thermodynamic instability of the solutions and hence phase separation. At high protein contents(60%)the protein rich phase produces deformed helical crystalline spherulites.By electron microscopy the protein rich phase contains predominantly oligomers of about 400 Å diameter, but no evidence for long range order could

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1974.tb02384.x

出版商:Blackwell Publishing Ltd    

年代:1974

数据来源: WILEY