Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.     

Development of continuous microwave-assisted protein digestion with immobilized enzyme

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC–MSⁿ. Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS–PAGE and LC–MSⁿ. Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC–MSⁿ profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.     

A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.     

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39. Other (62, 51, and 25 kD) quantitatively minor peripheral proteins also interact with IP39 on the nitrocellulose overlays, and the possible significance of this binding is discussed.     

重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中,构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1,转化大肠杆菌BL21（DE3）获得表达菌株,经IPTG诱导发酵的菌体超声破碎后,裂解液用Ni离子亲和层析纯化得到融合蛋白,经肠激酶裂解,再次Ni离子亲和层析,得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测,样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa,肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断,均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。     

Kinetic characterization of sequencing grade modified trypsin

Prior to analysis by mass spectrometry, protein samples are often digested. Maximizing the peptide yield from digestion can increase the number of peptides detected and the confidence in protein identification. To determine the optimal conditions for digestion, the Michaelis-Menten kinetic parameters for Promega sequencing grade modified trypsin were measured over a range of temperatures and pHs. The results indicate that an increase in digestion temperature above 37 degrees C, the temperature traditionally used in digestion methods, could offer an increase in peptides detected.     

Characterization of fragments produced by clostripain digestion of proteoglycans from the Swarm rat chondrosarcoma

Rat chondrosarcoma proteoglycan aggregate samples were digested with the protease clostripain (from Clostridium histolyticum) for various times. The progress of digestion was studied by Sepharose 2B chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After complete digestion, the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid was separated from the chondroitin sulfate-peptide clusters released by the enzyme. In this limit complex, the M_r of the link protein was 42,000, slightly smaller than the M_r of 45,000 observed for intact link protein. The chondroitin sulfate-peptides contained an average of about seven to eight polysaccharide chains per peptide and, after chondroitinase ABC digestion, were found to consist of two size classes of peptides. By comparison, chondroitin sulfate-peptides isolated from trypsin digests contained four to five chains per peptide and contained primarily the smaller size class of peptides. At early digestion times with clostripain, several distinct molecular weight intermediates containing hyaluronic acid-binding sites were identified on sodium dodecyl sulfate-polyacrylamide gels. These intermediates, with M_r, values of about 125,000, 100,000, and 85,000, decreased with increasing digestion time to yield a limit polypeptide (M_r = 67,000). Procedures are described for purifying this limit polypeptide and the link protein for further characterization. The results indicate that clostripain can be used to fragment proteoglycan molecules selectively to define different functional regions for study.     

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Five new expression vectors for recombinant human insulin production (pPT-B5Kpi, pPT-T10Rpi, pPT-T13Rpi, pPT-H27Rpi, pPT-B5Rpi), which have different sizes and leader peptide structure, were constructed and compared based on their expression level, yields of S-sulfonated preproinsulin (SSPPI) and folded proinsulin and enzymatic conversion rate. The ranking of expression level of the five fused proinsulins was H27R ? T10R > B5K > T13R ≈ B5R. In particular, the expression level of H27R was more than double (60-70%) the level of the other fused proinsulins, and this high expression level led to large amounts of SSPPI, folded proinsulin and insulin. Changes to the leader peptide structure affected not only protein expression level, but also refolding yield because the leader peptide affects protein conformation and hydrophobicity. The refolding yield of H27R was 85% at 500 L pilot scale. This high refolding yield was caused by the hydrophilic character of H27R. However, the β-mercaptoethanol concentration needed for refolding and the pH required to precipitate impurities after refolding had to be changed for high refolding yield. To avoid using CNBr, which is used to cleave fusion proteins, we used lysine and arginine linkers to connect the fusion protein and proinsulin. This fusion protein could be simultaneously cleaved by trypsin during enzymatic conversion to eliminate the C-peptide. The length and kind of leader peptide did not affect the enzyme reaction rate. Only the leader peptide linker connecting the B-chain influenced enzyme reaction rate. By testing several leader peptides, we constructed a new strain with 30% increased productivity based on expression level, refolding yield and enzyme reaction.     

Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine^B30 as a fusion protein with the S. cerevisiaeα-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine^B29 to glycine^A1, results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic–aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. Received: 8 February 2000 / Received revision: 2 May 2000 / Accepted: 2 May 2000     

High-yield production of recombinant endothelin-1.

A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.     

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases,using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Factors affecting the microbial digestion of an industrial seaweed-based residue

Commercial preparation of a seaweed extract from the brown alga Ascophyllum nodosum for use as fertiliser and soil improver produces a sludge residue which requires remediation. This residue is rich in nutrients and offers the potential for other value-added products. The residue composition was analysed, a microbial digestion process for the residue was developed, and several factors affecting the digestion process were studied. The residue showed an alkaline pH (8.61?±?0.39) and 16% (w/w) total solids, which comprised 40.6% mineral, 29.5% fibre, 24.3% lipid, 4.9% protein and 0.5% polyphenols. The optimised digestion system included a 3-day anaerobic phase to decrease pH (from 8.96?±?0.40 to 7.72?±?0.38), the addition of an inoculum, followed by a 10-day aerobic phase where the insoluble material was digested. Every 3 days, the solubilised material was decanted and replaced with water to delay metabolite inhibition. The rate of digestion (decrease in insoluble material of 28.6?±?14.2% over 13 days) was influenced by the initial insoluble (R ²?=?0.773) and soluble (R ²?=?0.672) matter, the pH at the beginning of the aerobic phase (R ²?=?0.528) and by the accumulation of solubilised digestion products. A compositional analysis of the insoluble material after digestion showed that the lipid content of the residue was 96% digested and that the proportion of protein increased by 82.4%. Inocula and metabolite inhibition were critical features of A. nodosum residue digestion. Similar organic residues require a carefully chosen inoculum and a minimum initial insoluble content (65–70%) and/or a maximum soluble content (25.30%).     

The insulin-directed phosphorylation site on ATP-citrate lyase is identical with the site phosphorylated by the cAMP-dependent protein kinase in vitro

32P-labeled ATP-citrate lyase isolated from 32P-labeled hepatocytes treated with insulin contained 1.6-1.8-fold greater 32P-radioactivity per mg protein than control enzyme. Both enzyme preparations were digested in parallel with trypsin until 94% of all 32P-radioactivity was rendered acid soluble. Quantitative high performance liquid chromatographic peptide mapping of the tryptic digests revealed a principal 32P-peptide which accounted for at least 80% of the insulin induced increment in 32P-radioactivity of native lyase. This peptide was purified, sequenced, and the site of 32P-phosphorylation assigned by two methods: electrophoresis (pH 6.5) of residual peptide after each step of Edman degradation and solid phase sequencing. The site of insulin-directed phosphorylation of ATP-citrate lyase (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg) is the same as that directed by glucagon, and, in turn, identical with that phosphorylated by the cAMP-dependent protein kinase in vitro.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.     

Identification of neighbor relationships among proteins in the 30 S ribosome: intermolecular cross-linkage of three proteins induced by tetranitromethane

Earlier studies have indicated that the reaction of tetranitromethane with the 30 S riboaome from Escherichia coli results in the disappearance of two protein bands from the polyacrylamide gel electrophoresis pattern (Craven et al., 1969b). As tetranitromethane is known to induce intermolecular cross-linkage in other protein systems, we studied further this reaction with the view that it might yield knowledge of protein-protein neighbor relationships within the ribosome.The use of two-dimensional polyacrylamide gel electrophoresis showed that the reaction with tetranitromethane caused the disappearance of four proteins from the pattern of 30 S ribosomal proteins. It was shown that this alteration in electrophoretic behavior was not due to simple protein modification (e.g. production of 3-nitrotyrosine), as reaction with extracted protein in 8 M-urea resulted in no observable change in the electrophoretic pattern.It was also shown that three of these proteins could be uniquely labeled with [¹⁴C]iodoacetate without changing their reactivity with tetranitromethane. Thus, ribosomes were labeled with [¹⁴C]iodoacetate, reacted with tetranitromethane and the radioactive reaction products were isolated by column chromatography and preparative gel electrophoresis. The radioactive peptide patterns of the three proteins digested by trypsin were compared with the three major reaction products. One of these products was shown to contain the radioactive tryptic peptides of all three proteins. We believe that this reaction product is an intermolecular cross-linked aggregate of these three proteins, identified as S11, S18 and S21. We suggest that these three proteins are clustered closely together in the 30 S ribosome. The fourth protein, S12, may also be involved in this aggregate.     

The peptides of chloroplast membranes: I. The soluble coupling factor (Ca2+-ATPase)

The chloroplast coupling factor (CF₁) was analyzed by gel electrophoresis in SDS and found to contain two major bands in equal amounts with mobilities corresponding to molecular weights of 62,000 and 57,000 and three minor bands of molecular weights 38,000, 21,000, and 14,000. The peptides were present in comparable amounts in many different preparations of the protein and, therefore, were thought not to be tightly bound contaminants. The interaction between these five peptides was shown to be noncovalent.Incubation of the enzyme with trypsin, under conditions which activate the latent ATPase, was found to cause selective digestion of the five peptides; the 62,000 M_r peptide was the most susceptible to digestion, while the 57,000 M_r peptide was most stable to trypsin. When chloroplast membranes were exposed to trypsin in the light to activate the postillumination Mg²⁺-dependent ATPase activity, EDTA extraction solubilized a protein fraction which contained the normal CF₁ peptide pattern. Also, the membranes, when solubilized and chromatographed on SDS-gels did not show the disappearance of any band.The ATPase activity of the protein was highly susceptible to ionic strength, being 50% inhibited by monovalent salts at a concentration of 0.05 m.     

Peptic and tryptic digestion of peptides and proteins monitored by capillary electrophoresis with contactless conductivity detection

The feasibility of monitoring the peptic and tryptic digestion of peptides and proteins with capillary electrophoresis using contactless conductivity detection was investigated. The peptide minigastrin I and the proteins cytochrome c from bovine heart, human serum albumin (HSA), myoglobin, and bovine serum albumin (BSA) were digested off-line with pepsin, and the resulting peptide and amino acid fragments were successfully separated and detected by conductivity measurement. Cytochrome c and myoglobin were also subjected to off-line cleavage with trypsin. On-line digestion using the electrophoretically mediated microanalysis (EMMA) approach was demonstrated with cytochrome c and apomyoglobin using trypsin.