Effect of the insulin iodination on the reactivity of the inter-chain disulphide bonds towards sodium sulphite

Insulin iodination interferes with the ability of the interchain S.S bonds to react with sulphite at pH7. In insulin samples containing more than 5 iodine atoms/monomer unit, only one S.S bond/molecule reacts. The effect must be related to the substitution of the iodine into the tyrosyl groups, which probably causes a conformational rearrangement resulting in a steric hindrance of one of the interchain S.S bonds. The effect is removed by increasing the pH or by adding urea (8m) to the reaction mixture. The unreactivity of the S.S bond and the biological inactivation occur at the same ;critical' iodination level, suggesting that a same primary alteration of the molecule is responsible for both the effects.     

The reactivity of the disulphide bonds of purified proteins in relationship to primary structure

1. With the aid of a coupled system involving glutathione reductase, the reaction of glutathione with the disulphide bonds of purified proteins has been studied. 2. Bovine serum albumin, conalbumin, lysozyme, trypsin inhibitors from egg white, lima bean and soya bean either did not react with glutathione or reacted only slightly. With these proteins reactivity was markedly increased by limited proteolysis. 3. Bovine and human gamma-globulins, fibrinogen and beta-lactoglobulin exhibited some reactivity (less than 15%) with glutathione and again this was increased by limited proteolysis. Pepsin, trypsin and chymotrypsin exhibited greater reactivity than the proteins previously mentioned. Di-isopropylphosphoryl-chymotrypsin exhibited less reactivity than chymotrypsin, suggesting that autolysis under the experimental conditions used contributed towards the reactivity of this protein. Proteolysis also increased the reactivity of these proteins. The three disulphide bonds of insulin were reduced by glutathione. 4. Above 35 degrees the disulphide bonds of serum albumin show a progressive increase in reactivity and at 55 degrees half of the bonds become accessible to glutathione. 5. From the results obtained with the proteins investigated, the conclusion reached is that the disulphide bonds of native proteins are structurally protected and do not react with glutathione under physiological conditions.     

Putative disulfide-forming pathway of porcine insulin precursor during its refolding in vitro

Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.     

The reactivity of the disulfide bonds of human serum haemopexin

The reactivity of the disulfide bonds of the specific haeme-binding plasma protein-human haemopexin has been studied with 2-mercaptoethanol. A molecule of haemopexin has six intrachain disulfide bridges (Takahashi et al., 1985) or which four are reactive while the remaining two can be reduced in the presence of greater than or equal to 4M urea. Disruption of the four reactive disulfide bonds in apohaemopexin abolishes the haeme binding ability. In equimolar haeme-haemopexin complex only one disulfide is reactive which suggests a large change in the tertiary structure of this protein on haeme binding.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

The insulin A and B chains contain structural information for the formation of the native molecule. Studies with protein disulphide-isomerase.

It has been shown previously [Tang, Wang & Tsou (1988) Biochem. J. 255, 451-455] that, under appropriate conditions, native insulin can be obtained from scrambled insulin or the S-sulphonates of the chains with a yield of 25-30%, together with reaction products containing the separated A and B chains. The native hormone is by far the predominant product among the isomers containing both chains. It is now shown that the presence of added C peptide has no appreciable effect on the yield of native insulin. At higher temperatures the content of the native hormone decreases whereas those of the separated chains increase, and in no case was scrambled insulin containing both chains the predominant product in the absence of denaturants. Both the scrambling and the unscrambling reactions give similar h.p.l.c. profiles for the products. Under similar conditions cross-linked insulin with native disulphide linkages can be obtained from the scrambled molecule or from the S-sulphonate derivative with yields of 50% and 75% respectively at 4 degrees C, and with a dilute solution of the hexa-S-sulphonate yields better than 90% can be obtained. The regenerated product is shown to have the native disulphide bridges by treatment with CNBr to give insulin and by the identity of the h.p.l.c. profile of its peptic hydrolysate with that for cross-linked insulin. It appears that the insulin A and B chains contain sufficient information for the formation of the native molecule and that the role of the connecting C peptide is to bring and to keep the two chains together.     

Heterogeneity of ovine prolactin after labeling with iodine 125: value of labeling with lactoperoxidase]

The authors have shown an heterogeneity of the ovine prolactine molecule after labelling with iodine 125. As well with chloramine T as with lactoperoxydase, it appears three molecular species which react with the immune serum antiprolactine (I.S.). The first species is of high molecular weight and is probably constituted of aggregates. Their combination with the I.S. is non specific and give blanks of high value. The second species is a dimere and the third one is the monomere. The two last species react with the I.S. and can give competition curves when they are choosen as tracer. However, if one uses as tracer a product obtained by labelling with chloramine T, the competition appears for high concentrations of native hormone. As if the I.S. recognizes much more the labelled protein than the native one. But if one uses the same species but labelled with lactoperoxydase, the competition appears for concentrations lower than five nanogrammes. In the same time one can see that the specificity toward different I.S. is modified. The authors think that the labelling with lactoperoxydase better preserves the tertiary structure of the native protein than do the labelling with chloramine T.     

Accessibility of phosphodiester bonds in the yeast ribosomal 5 S RNA protein complex

The tertiary structure of the protein-associated yeast ribosomal 5 S RNA was examined using ethylnitrosourea reactivity as a probe for phosphodiester bonds. A reduced reactivity was consistently observed in at least nine residues within four distinct regions of the RNA sequence. Seven of these were also observed in three regions of the free RNA molecule while two, A27 and G30, were only present in the ribonucleoprotein complex. The results strongly suggest that the tertiary structure of the free eukaryotic 5 S RNA is largely conserved in the 5 S RNA-protein complex although it appears to be further stabilized in interaction with the ribosomal protein.     

Proteins can convert to beta-sheet in single crystals

Raman microscopy was used to follow conformational changes in single protein crystals. Crystals of native insulin and of the 5S and 12S subunits of the enzyme transcarboxylase showed a mixture of Raman marker bands signifying alpha-helix, beta-sheet and nonordered secondary structure. However, by reducing the S-S bonds in the insulin crystal, or by lowering the pH for the 5S crystal, or by soaking substrates into the 12S crystal, the secondary structure in each crystal became predominantly beta-sheet. The beta-form crystals could be dissolved only with difficulty and yielded high-molecular weight protein aggregates, indicating that the beta-sheet formation involves intermolecular contacts. Although their morphology appeared unchanged, the crystals no longer diffracted X-rays. Using crystals that had not been exposed to laser light, the dye thioflavin T formed highly fluorescent complexes with the "beta-transformed" crystals but not with the native crystals.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

Identification and partial characterization of multiple native and neoantigenic epitopes of human C-reactive protein by using monoclonal antibodies

Multiple mAb to human C-reactive protein (CRP) were prepared which reacted preferentially with either native CRP, modified CRP (expressing "neo-CRP" determinants) or both forms of the molecule. These mAb were divided into four groups according to their binding characteristics to various CRP preparations and CRP peptides by using a combination of ELISA, dot blot, and Western blot assays; they were further characterized based upon their reactivity with CRP in the presence of calcium and inhibition by phosphorylcholine. The first group consisted of mAb that reacted only with native CRP, and served to define four distinct native CRP epitopes. The second group consisted of mAb that reacted with native CRP and also with CRP modified by direct immobilization on polystyrene plates, urea-chelation or SDS treatment in the absence of calcium, thus identifying a fifth native CRP epitope; these mAb displayed significantly greater reactivity with native than with modified CRP. The third group included mAb that reacted only with modified CRP and with the larger amino-terminal fragment (residues 1-146) of pronase-cleaved CRP. The fourth group included mAb that reacted only with modified CRP and with the smaller carboxyl-terminal fragment (residues 147-206) of pronase-cleaved CRP; most of these antibodies also reacted with the carboxyl-terminal octapeptide (residues 199-206) of CRP. These experiments have identified mAb that react preferentially with distinct conformational and sequence-determined epitopes of native and modified forms of the CRP molecule, respectively; provide partial identification of the epitopes with which they interact; point to the presence of at least five epitopes on native CRP and at least three epitopes on modified CRP; and provide antibodies suitable for identification and quantitation of native and modified forms of CRP. The mAb directed against neo-CRP epitopes may help identify the presence of this pentraxin and antigenically-related proteins at previously unappreciated sites.     

Reactive properties of the organic solvent-soluble lipase

In a previous report, the organic solvent-soluble lipase was prepared using a synthetic detergent, didodecyl glucosyl glutamate, and it was estimated that 150 +/- 30 molecules of the detergent were attached to one lipase molecule based on gel permeation chromatography and chemical analysis. In this paper, the reactivity of the organic solvent-soluble lipase was compared with that of the native lipase to study the effect of the surrounding detergent on the thermostability and enzymatic reactivity. The activity of the organic solvent-soluble lipase was preserved in the organic solvents up to a temperature of 50 degrees C as in the case of the native lipase in buffer (pH 7.0). The influence of the chain length of fatty acids of the substrate triacylglycerols on the hydrolysis activities was studied. The organic solvent-soluble lipase hydrolyzed triacylglycerols with longer chains more rapidly than the native lipase. The presence of Ca2+ at 0.1 mM stimulated the activity of the native lipase, whereas Ca2+ at a high concentration inhibited it. On the other hand, even at a low concentration, Ca2+ inhibited the activity of the organic solvent-soluble lipase. These results suggest that the detergent attached to the lipase molecule affected the reactive properties.     

The substrate specificity of thermomycolase, an extracellular serine proteinase from the thermophilic fungus Malbranchea pulchella var. sulfurea.

The specificity of thermomycolase toward glucagon and the oxidized A and B chains of insulin was investigated. Extensive digestion of glucagon occurred when conducted at pH 7.0 and 45 degrees C for 40 min, whereas hydrolysis of only three peptide bonds occurred at pH 7.0 and 28 degrees C for 5 min. A similar situation was observed for the oxidized B chain of insulin, which exhibited only a single major cleavage after 5 min at 25 degrees C. No well-defined specificity for particular amino acid residues was evident, but ready hydrolysis of peptide bonds occurred within sequences containing non-polar residues. This endoproteinase must therefore possess an extended hydrophobic binding site for polypeptides. Thermomycolase hydrolysed acetylalanylalanylalanine methyl ester and elastin-Congo Red at 22 and 8.5 times the rate of porcine elastase respectively. A limited degradation of native collagen and significant hydrolysis of benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro were suggestive of some collagenase-like activity. No keratinase activity was apparent.     

Conformation of beta 2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond

Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.     

Specificity of the collagenase from the insect Hypoderma lineatum

Specificity of the collagenase from the larvae Hypoderma lineatum, a serine protease related to trypsin, has been investigated by using native collagen and non-collagenous substrates. At 25 degrees C and neutral pH the degradation of collagen by the larval enzyme in solution results in a 52% loss of specific viscosity, without loss of helicity. Electron microscopy of segment-long-spacing crystallites of the digest shows the occurrence of one cleavage region between bands 41 and 44 whereas Edman degradation indicates several cleavage loci in this region. Hypoderma collagenase differs from proteinases I and II from the crab Uca pugilator, which catalyse cleavages in multiple regions of the collagen molecule, and also from vertebrate collagenases, which cleave collagen only between residues 775 and 776. Apart of specific action on collagen, Hypoderma collagenase degrades the oxidized chain B of insulin; the major cleavage occurs at the Leu15-Tyr16 bond followed by two minor cleavages at the Arg22-Gly23 and Lys29-Ala30 bonds. The larval enzyme has no action on synthetic peptide substrates of trypsin or chymotrypsin.     

Reinvestigation of the sulfhydryl reactivity in bovine brain S100b (beta beta) protein and the microtubule-associated tau proteins. Ca2+ stimulates disulfide cross-linking between the S100b beta-subunit and the microtubule-associated tau(2) protein

Zn2+ and Ca2+ affect the conformation of bovine brain S100b (beta beta) protein and the exposure of its Cys-84 beta. Zn2+ binding to high-affinity sites of native S100b protected the sulfhydryl groups against the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoate) and antagonized the Ca2+-stimulated reactivity of Cys-84 beta toward the reagent. Spectroscopic studies on the fluorescence properties of labeled S100b with the fluorescent probes bimane and acrylodan at Cys-84 beta confirmed the antagonistic effect of Ca2+ and Zn2+ with respect to the conformational properties of the protein. Measurements of fluorescence dynamics on bimane-labeled S100b indicated that the slow monomer-dimer equilibrium that characterizes the apoprotein at micromolar concentrations was shifted to the monomer form in the presence of Zn2+, a fact that could explain the previously reported Zn2+-dependent increase of S100b protein affinity for calcium. The difference in the effects of Ca2+ and Zn2+ on the reactivity of Cys-84 beta in S100b was confirmed when we observed that Ca2+ and Zn2+ have opposite actions on the formation of disulfide bridges between Cys-84 beta of the S100b beta-subunit and sulfhydryl groups on the microtubule-associated tau(2) protein. Ca2+ stimulated the covalent complex formation whereas Zn2+ inhibited it. We suggest that Zn2+ may have a modulatory function on Cys-84 beta reactivity in the S100b beta-subunit in vivo. Two types of divalent complexes between tau(2) and beta-subunit were formed in the presence of Ca2+, an equimolar complex tau(2)-beta 1 and a complex of one molecule of tau(2) with two beta-subunits, tau(2)-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of iodination on the distribution of peptide hormones in aqueous two-phase polymer systems

1. The effect of iodination on the distribution of peptide hormones into the aqueous two-phase dextran-polyethylene glycol system and on the solubility of these hormones in aqueous polyethylene glycol and in water was assessed. Hormones that were studied included insulin, glucagon and parathyroid hormone. 2. The partition coefficient of native insulin in the dextran-polyethylene glycol system showed a minimum (about 1) near the isoelectric point of the hormone (pH 5). Partial iodination of insulin (one atom per molecule) caused little change in the distribution of the hormone. More extensive iodination markedly decreased the partition coefficient in the region of the isoelectric point and displaced the pH value at which the partition coefficient was a minimum towards lower values. 3. The solubility of native insulin in aqueous polyethylene glycol and in water showed a pH-dependence similar to that observed for the distribution in the dextran-polyethylene glycol system. Iodination of insulin decreased the solubility of the hormone in polyethylene glycol and in water in parallel, and decreased the pH value at which solubility was a minimum. The changes in solubility correlated with the degree of iodination and accounted for the changes in distribution observed at high concentrations of insulin. 4. Comparable effects of iodination on distribution and solubility were also observed with glucagon. 5. At concentrations of insulin below its maximum solubility, serum proteins caused a decrease in the partition coefficient of iodinated hormone, but not of native hormone. These effects correlated with the degree of iodination and resulted from a co-precipitation of iodinated insulin with serum proteins.     

Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.