Isolation and characterization of the cyanogen bromide peptides from the B fragment of diphtheria toxin

Homogeneous fragment B, obtained through nicking of diphtheria toxin with insoluble trypsin, was cleaved with cyanogen bromide in 70% formic acid. After citraconylation, the cleavage products were separated by gel filtration on Sephadex G--75 and purified by gel filtration, ion-exchange and thin-layer or paper chromatography. Six CNBr peptides were characterized, the composition of which account for the total amino acid content of fragment B. Their apparent molecular weights are: CB 1, 12 000; CB 2, 14 000; CB 3, 8000; CB 4a, 2400; CB 4b, 2200; CB 5, 2200. CB 4a is the NH2--terminal peptide; it contains the cysteine residue of the disulfide bridge linking fragment B to fragment A. CB 3 is the COOH--terminal peptide; it bears the disulfide bridge of fragment B. Characterization of two CNBr--derived overlapping peptides provided the positioning of CB 4b and CB 2 and allowed an alignment of the CNBr peptides of fragment B to be proposed.     

Isolation and characterization of free radical scavenging activities peptides derived from casein

A peptide having the strong free radical scavenging activities was separated from casein protein hydrolysate by chromatographic analyses such as ion-exchange and gel filtration. SP-II fraction obtained by SP-Sephadex C-25 chromatography showed the most potent superoxide anion scavenging activity (SOSA), and it was further separated into a peptide using an octadecylsilano-high performance liquid chromatography. The amino acid sequence of the peptide was Tyr-Phe-Tyr-Pro-Glu-Leu (YFYPEL). The concentration of the test compound required to reduce the produced superoxide anion to one-half (IC(50)) value for SOSA was 79.2 microM using tetrazolium salt 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate method. The IC50 value for the 1,1-diphenyl-2-picrylhydrazyl radical and hydroxyl radical scavenging activities were 98 and 251 microM, respectively, based on the electron spin resonance method. We characterized SOSA of the C-terminal sequence using EL, PEL, YPEL, and FYPEL. The activities preferred sequences were EL>YFYPEL>FYPEL>YPEL>PEL, suggesting that the Glu-Leu sequence is important for the activity.     

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.     

Solution conformations of the N-terminal CNBr fragment of glycogen phosphorylase and its interaction with calmodulin

The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase.     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils

Cathelicidins are a family of gene-encoded antimicrobial peptides found in mammals. Seven cathelicidin genes have been identified in sheep, but up to now only two variants of one of these predicted peptides (OaBac5) have been purified from ovine neutrophils. In this work numerous proline/arginine-rich cathelicidin peptides were purified, including the originally predicted OaBac5 and another OaBac5 variant. As well as this, the C-terminus of the predicted OaBac7.5 and various truncated forms of OaBac11 were purified. Even though these peptides were much smaller than those predicted, they still displayed antimicrobial activity.     

Isolation and characterization of proSS1-32, a peptide derived from the N-terminal region of porcine preprosomatostatin

A peptide derived from the N-terminal region of porcine prosomatostatin, proSS1-32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L. This sequence obviously comprises residues 1-32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu-Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.     

Mapping of antigenic domains on phytochrome from etiolated Avena sativa L. by immunoblot analysis of proteolytically derived peptides

Several monoclonal antibodies to phytochrome that interact with putative functionally important domains have been previously identified. The locations of some of these domains are determined here by epitope mapping experiments that utilize immunoblot analyses of proteolytically degraded phytochrome. Seven independent epitopes are identified. An epitope that is recognized by monoclonal antibody Oat-25 is confirmed to be wholly located near the N terminus of phytochrome. This domain undergoes a conformational change when phytochrome is interconverted between its red- and far-red-absorbing forms and is recognized by Oat-25 better in the red-absorbing form. A second domain that also undergoes a photointerconvertible conformation change and that contains the epitope for Oat-16 is localized near the site of chromophore attachment, which is about 36 kDa from the N terminus. A third domain, which contains the most highly conserved epitope on phytochrome that has so far been identified, is recognized by Pea-25 and is located about 85 kDa from the N terminus. Other epitopes and their approximate distances from the N terminus are those recognized by Oat-22 (36 kDa), Oat-13 (65 kDa), and Oat-8 and Oat-28 (70-75 kDa). Even though epitopes for Oat-16 and Oat-22, as well as for Oat-8 and Oat-28, are close together, competitive binding assays indicate that they are different. Immunoblot analyses also indicate that the epitope for Oat-28 is further from the N terminus of phytochrome than is that for Oat-8.     

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical characterization

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.