Polypeptide Chain Of Molecular Weight Research Articles

Dissolution and regeneration of wool keratin in the deep eutectic solvent of choline chloride-urea

Wool keratin is a kind of renewable protein which has great potential in environment-friendly materials. In this paper, the dissolution of wool fiber and the regeneration of wool keratin in the deep eutectic solvent (DES) of choline chloride-urea was detailed studied. The results showed that 35.1 mg wool completely dissolved in 1 g DES at 130 °C in 5 h. The polarizing microscope, scanning electron microscope (SEM) images along with the amino acid analysis suggested that the DES mainly dissolved the wool cortex layer. Compared with the raw wool, the regenerated wool keratin contained much more fragments of low molecular weight polypeptide chains from SDS-PAGE results. And the percentage of α-helix dramatically decreased, while the content of β-sheet and disordered structure increased. All the results indicated that the DES can be used as a kind of green solvent for the regeneration of wool keratin without losing the long peptide chains. The method also offered a possibility of large-scale for wasted wool reuse.

Modified extraction and characterization of keratin from Indian goat hoof: A biocompatible biomaterial for tissue regenerative applications

ABSTRACTThe Goat Hoof Keratin (GHK) is a valuable biodegradable substance for making tissue regeneration. The protein substance from GHK powder is extracted in a clean form by using modified shindai solution technique. Electrophoreical analysis produces polypeptide chains of molecular weight of 40–45 kDa using SDS-PAGE method. FTIR spectra show the characteristic absorption band for α-helix and β-sheet peptide bonds. DSC analysis provides the GHK denaturation occurred at temperature ranging from 200–250°C and thermal degradation occurs at 211°C & 387°C using TGA. The biocompatibility of GHK examined using MTT (3(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay of fibroblast cells shows 91% cell viability.

Yield and Gel Strength of Gelatin Extracted from Smoked Salmon (Salmo salar) Skins

ABSTRACTThe gelatin yield and gel strength of gelatin extracted from the skins of smoked salmon were determined. The skins had a crude protein content of 43.5 ± 1.2% and an estimated collagen content of 23.6 ± 1.0%. Following an alkali extraction process under varying extraction conditions, the protein content varied from 2.78% to 32.1%, and the gelatin yield ranged from 2.23% to 22.4% of the initial skin weight. The gelatin purity (gelatin/protein) ranged from 55.4% to 100%, with the highest Hyp/protein sample arbitrarily designated as being 100% pure, i.e., an assumption of pure gelatin. Statistical analysis showed that HCl concentration, pretreatment temperature, and extraction temperature significantly affected the protein yield and gelatin yield. The wide range of gel strengths indicates that the gelatin might have been hydrolyzed to different extents during the different extraction and cooling processes. Statistical analysis did not show that any of the factors studied affected the gel strength. However, the data indicated that low NaOH concentrations and HCl concentrations might not prevent hydrolysis during the extraction process. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) showed that the gelatins contained α- and β-chains and that the gelatins composed of higher concentrations of larger molecular weight polypeptide chains had higher gel strengths.

Extraction and characterization of keratin from bovine hoof: A potential material for biomedical applications.

Keratin from the hoof is a less explored source for making valuable products. In this paper we present the extraction of pure keratin from bovine hooves and characterized them to better address the possible exploitation of this bio-resource as an alternative material for tissue engineering applications. The keratin protein from the pulverized hooves was extracted by reduction, which was observed to be pure, and two polypeptide chains of molecular weight in the range of 45–50 and 55–60 KDa were determined using SDS-PAGE assay. FTIR analysis complementing circular dichroism (CD) data, established that hoof keratin predominantly adopted α-helical conformation with admixture of β-sheet. The keratin was shown to have appreciably high denaturation temperature (215°C) as indicated by differential scanning calorimetric (DSC) analysis. Thermogravimetric analysis (TGA) also showed the retention of 50% of the original weight of the sample even at a temperature of 346°C. The keratin from the hoof had been observed to be biocompatible when analyzed with MTT assay using fibroblast cells, showing more than 90% cell viability. Hence, hoof keratin would be useful for high value biomedical applications.

Purification and characterization of a β-glucan binding protein from the haemolymph of freshwater prawnMacrobrachium rosenbergii

β-glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β-glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β-glucan increased the specific PO activity compared with that of β-glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS-PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS-PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non-covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti-βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL−1. Furthermore, the applicability of the developed ELISA is discussed.

“9 + 0” Immotile Spermatozoa in an Infertile Man*

In this paper the peculiar case of an infertile man, possessing only normally shaped, but stiff and immotile spermatozoa is described. All the sperm are conventionally structured, with the constant characteristic of the absence of central tubules and projections forming the so-called central sheath. Electrophoretic analysis of the high molecular weight polypeptide chains attributed to dyneins shows the constant absence of one chain. The importance of the central structure, usually belonging to the "9 + 2" model spermatozoa, and the possible localization of a dynein chain in this region are discussed.

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5–6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy ( E a) was found to be 44.0 ± 0.3 kcal mol −1. The activation enthalpy (Δ H ∗), free energy change (Δ G ∗) and entropy (Δ S ∗) were estimated to be 43 ± 4 kcal mol −1, −26 ± 3 kcal mol −1 and 204 ± 10 cal mol −1 K −1, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P 1 position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.

Haemorrhagic protein of Russell's viper venom with fibrinolytic and esterolytic activities

A haemorrhagic toxin specifically active on skin and muscle at the site of introduction in mice has been purified from Vipera russelli russelli (Indian subspecies of Russell's viper) venom by CM-Sephadex C-50 ion exchange chromatography and size exclusion (SE)-HPLC. This toxic protein also has strong fibrinolytic and arginine esterolytic activities. The purified preparation was a single polypeptide chain of molecular weight 73,000, as revealed by SDS-PAGE and SE-HPLC under native and denatured conditions. It has been named as VRR-73. Atomic absorption spectrometry indicated the existence of Mg 2+ in a mol per mol ratio. Antiserum was effective in neutralizing haemorrhage when administered immediately following VRR-73 but was ineffective in inhibiting fibrinolytic and esterolytic activities. On the other hand phenylmethyl sulphonyl fluoride and EDTA inhibited fibrinolysis and esterolysis but did not affect haemorrhage. Thermal denaturation of VRR-73, after exposure at 100°C for 10 min, led to inactivation of all of its activities. Fibrinolytic and esterolytic activities, but not the haemorrhagic activity, were slowly regained after cooling at 25°C. Thus the two pathological activities of VRR-73 appear to be associated with two different regions of the molecule.

Preparative electrophoresis on linear polyacrylamide‐agarose composite gels

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

Purification and Partial Characterization of an Induced Antibacterial Protein in the Silkworm, Bombyx mori

Injection of live Escherichia coli into larvae of the silkworm, Bombyx mori, induces antibacterial activity in the hemolymph. The major induced antibacterial activity was purified in two steps by CM-Sephadex C-50 and Sephadex G-100 column chromatography. After trypsin treatment, the purified antibacterial protein lost its activity and the antibacterial activity was found to be partially heat labile. The purified protein was a single polypeptide chain of molecular weight 16 kDa. The 20 N-terminal amino acid sequence of the protein was determined and this sequence showed homology with the N-terminal amino acid sequence of lysozymes reported in other species. The purified protein was found to have comparable antibacterial activity against both E. coli and Micrococcus luteus. The purification of antibacterial protein and the antibacterial properties of the purified protein are discussed.

Characterization of homogeneous atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

We previously reported the discovery and partial characterization of bovine atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor, which is associated with atrial granule membranes. We now report the physicochemical properties of electrophoretically homogeneous enzyme purified by a series of chromatography steps from a subcellular fraction enriched for atrial granules. The enzyme tends to associate during purification to higher molecular weight species, but SDS-PAGE analysis reveals a single polypeptide chain of molecular weight 70,000. The enzyme is activated 2–3 fold by Ca +2 and 1.5-fold by Mg +2 and is nearly 100% inhibited by Zn +2 or Co +2. Thus, the enzyme can be considered a calcium activated, neutral pH, serine proteinase. Based on the hydrolysis of numerous synthetic peptide substrates, the recognition sequence for the enzyme within the pro-hormone has been mapped to A 96PRSLRR 102; cleavage occurs at the Arh 98-Ser 99 bond yielding bioactive atrial natriuretic peptide directly from the pro-hormone. The doublet of basic amino acids is part of the recognition sequence but is not the primary cleavage site. It is our hypothesis that the processing site sequence acts as a recognition element for the endoproteinase and resides at the surface of the pro-hormone and thus contributes to the molecular basis for limited proteolysis.

Initial binding of 2‘-deoxynucleoside 5‘-triphosphates to human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.

Analysis of the mechanism of interaction of simian Ku protein with DNA

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.

The α-bungarotoxin receptor purified from a human neuroblastoma cell line: Biochemical and immunological characterization

The pharmacological and electrophysiological characteristics of the α-bungarotoxin receptor present on the human neuroblastoma cell line IMR-32 indicate that this receptor is not associated with an acetylcholine-operated ionic channel. In this paper we report its biochemical purification and immunological characterization. This molecule has a standard sedimentation coefficient of 10S and sodium dodecyl-sulphate gel electrophoresis shows that it is made up of three polypeptide chains of molecular weights of 67,000, 60,000 and 52,000. Ligand binding to blots of purified receptor revealed that only the polypeptide of molecular weight 52,000 is bound by [ 125I]α-bungarotoxin . The purified α-bungarotoxin receptor was bound by polyclonal antibodies raised against purified fetal calf, Torpedo and chick optic lobe nicotinic receptors and by the sera of myasthenie patients. Furthermore, despite the fact that a number of different immunological techniques were used, it was impossible to label this α-bungarotoxin receptor with mAb 35, a monoclonal antibody which binds some neuronal nicotinic receptors. Rabbit antisera against the purified α-bungarotoxin receptor were used to compare this protein with other known nicotinic receptors and, once again, it was demonstrated that there is some immunological cross-reactivity between the α-bungarotoxin receptor present on neuroblastoma cells and Torpedo, fetal calf and chick optic lobe nicotinic receptors. All these immunological data, together with previously published pharmacological and molecular biology data, demonstrate that the α-bungarotoxin receptor present in nerve cells is neither a muscular nor a neuronal nicotinic receptor, although it has similarities with both.

Antigenicity of rat erythrocyte glycophorins

The relationship between rat red blood cell (RBC) glycophorins and the antigens recognised by anti-rat RBC antibodies was examined. Initially, murine monoclonal antibodies specific for surface epitopes on whole rat RBCs were tested for their reactivity with RBC membranes on Western blots and two were found which reacted with blotted antigens. These antibodies recognised two bands corresponding to the major PAS-stainable bands of rat RBC membranes (i.e., the glycophorins) and a number of minor bands, thus demonstrating that the bands are antigenically related. This band-pattern was remarkably similar to that obtained with mouse anti-rat RBC serum. Digestion with neuraminidase altered the electrophoretic mobility of most of the bands, providing additional evidence that they are sialoglycoproteins, although sialic acid was shown not to contribute to their antigenicity. The glycophorin nature of the major antigens was verified by reelectrophoresis and blotting of bands excised from SDS gels, which showed that they were interconvertible monomeric and dimeric forms of the same polypeptide chain. It is suggested that rat RBC glycophorins are a related family of sialoglycoproteins with the high molecular weight members being formed by dimerization of five lower molecular weight poly-peptide chains in various combinations.

Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking.

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.

Isolation, purification and some properties of a lectin from the winged bean (Psophocarpus tetragonolobus)

We have isolated by affinity chromatography a lectin from the seeds of the winged bean (Psophocrapus tetragonolobus) which agglutinated human (group A, B and O), sheep and rabbit, but not mouse erythrocytes. A molecular weight of 41,000 was obtained from gel filtration, and on sodium dodecyl sulphate polyacrylamide gel electrophoresis a single polypeptide chain of molecular weight 35,000 was seen both before and after reduction. Isoelectric focussing of the lectin on polyacrylamide gel gave a single band with a calculated isoelectric point of 4.0. The lectin was found to be rich in acidic amino acids; cysteine was not detected. Carbohydrate analysis revealed no covalently bound sugars.

Determination of polypeptide chain molecular weights of human and bovine band 3 protein from erythrocyte membranes by low-angle laser light scattering combined with high-performance gel chromatography in the presence of sodium dodecyl sulfate

Polypeptide chain molecular weights of human and bovine band 3 proteins which are glycoproteins of the erythrocyte membrane were determined as 101 000 ± 2000 for the former and 107 000 ± 2000 for the latter by using the low-angle laser light scattering technique combined with a high-performance gel chromatography column, an ultraviolet spectrophotometer and a differential refractometer in the presence of sodium dodecyl sulfate. The advantage of this method is that, unlike the sedimentation equilibrium technique, neither information on the binding to proteins of all ligands present nor the partial specific volume is required to evaluate the polypeptide chain molecular weight of proteins in a multicomponent system.

Probing the Nucleotide Binding Sites of Sarcoplasmic Reticulum Atpase by Photoaffinity Labeling

The Ca-ATPase of sarcoplasmic reticulum is a membranebound enzyme consisting of a single polypeptide chain of molecular weight 100,000-120,000 (1). The partial reactions of the catalytic and transport cycles of the enzyme have been extensively studied (2). However, the functional unit of the ATPase has not yet been clearly understood (1). The enzyme displays a nonMichaelian behavior for the ATPase activity with respect to Mg ATP, which has been attributed to an effect of ATP at low affinity regulatory sites (3-6). Alternatively, a half-of-the-sites reactivity mechanism has been suggested to explain the modulation of the turn-over rate by ATP (7, 8) and a possible interaction of subunits has been implicated (9, 10). As the stoichiometry of nucleotide binding sites of the enzyme is still controversial, it is difficult at present to decide which is the most likely mechanism. Photoaffinity labeling is an advantageous technique to clarify the stoichiometry and/or the topography of ATP binding sites (11), as it allows covalent attachment of nucleotides at possible regulatory sites. In the sarcoplasmic reticulum ATPase the putative regulatory sites cannot be titrated by conventional binding techniques because of their low affinity (12). Recently, Cable and Briggs (13) reported the photoaffinity labeling of a small fraction of the Ca-ATPase high-affinity catalytic sites by benzoylbenzoic ATP. We have been involved in the characterization of high and low affinity classes of nucleotide binding sites using fluorescent (14) and photoactivatable (15) ATP analogs. The fluorescent analog 3'-trinitrophenyl-ATP (TNP-ATP) revealed the existence of two classes of sites (14) but the fluorescence intensities measured did not permit the evaluation of the stoichiometry. In addition, TNP-ATP is not a substrate for the Ca-ATPase in that it is not hydrolyzed by the enzyme (14). To investigate the stoichiometry of the catalytic and regulatory nucleotide sites in the Ca-ATPase we have chosen to use ATP analogs which are photactivatable and could covalently attach to the enzyme upon illumination. Both UTP and 3'-arylazido-ATP were used. The irradiation of a mixture containing ATPase and 1 mM [3H] UTP resulted in a time-dependent incorporation ofUTP to the protein. The presence of nonradioactive ATP in the irradiation mixture prevented the photoincorporation of radioactive UTP; competition experiments performed with the ATP analogue TNP-ATP displayed similar protection.

S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization.

S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.