Proteolytic Activity Of Papain Research Articles

In vitro effect on the proteolytic activity of papain with proteins of the skin as substrate.

This work aims to evaluate the effects of enzyme concentration, pH, temperature and time course degree of hydrolysis (DH) of papain regarding further development of pharmaceutical and cosmetic formulations. The hydrolysis of casein, collagen, keratin and porcine skin at pH and temperature ranges of the human skin was evaluated. Also, low contact times of enzyme-substrate were studied. The incorporation of 3mM of cysteine improved the caseinolytic (PU), collagenolytic (CU) and keratinolytic (KU) activities of papain. In general, the increase from 0.1 to 1.0 or 2.0mgmL-1 of papain improved PU, CU and KU. When 2.0mgmL-1 of papain was used, the highest DH of casein, collagen and keratin was obtained at 240min (14%, 35% and 6% respectively). The decrease in pH and temperature reduced all proteolytic activities, but papain maintained at least 50% and 40% of its activity at 26°C and pH 4.5 respectively. Scanning electron micrographs of the surface of the skin showed that papain application had exfoliating activity. This preformulation study demonstrated that papain concentration, time of application and pH of the product should be evaluated when developing a product to promote the hydrolysis of the proteins of the skin.

Preferential and competitive role of hydrophilic/hydrophobic interactions quantifying amino acid-based ILs for papain stabilization

A plethora of amino acid based ionic liquid (AAIL) has been synthesized recently in order to thermally and structurally stabilize the proteins, however, critical impact of AAIL cations and anions hydrophilicity has been significantly ignored and subsided. Herein, the pivotal role of cations competitive hydrophilicity has been envisaged by investigating the proteolytic activity, structural stability, and alteration in microenvironment of papain in the presence of varying concentration of cholinium tryptophan ionic liquids [CHO][Trp]IL and tetraethylammonium tryptophan ionic liquids [TEA][Trp]IL. The proteolytic activity of papain in increasing concentration of [CHO][Trp]IL and [TEA][Trp]IL shows that at lower concentration of [CHO][Trp]IL more hydrophilic cholinium cation enhanced the papain activity from 100 to 131 %, however detrimental effects on the activity have been observed at lower concentration of [TEA][Trp]IL due to less hydrophilic nature of tetraethylammonium anions. Moreover, the highly interactive nature of [TEA][Trp]IL can be concluded by Fourier transmission infrared spectroscopy (FTIR). The UV–vis, steady state fluorescence and circular dichroism results also concluded the prowess of AAILs hydrophilic cation for stabilization nature. However, at higher concentration detrimental effects have been observed even for AAILs which are having hydrophilic cations. Thus, the study highlighted that [CHO][Trp]IL can enhance the activity and structure stability of papain more significantly while maintaining its surrounding microenvironment due to more hydrophilic nature of cholinium cation in contrast to [TEA][Trp]IL which consists of less hydrophilic tetraethylammonium cation. Moreover, with increase in the concentration of both AAILs, detrimental effects on the proteolytic activity and structural stability of papain have been observed. To differentiate further between the polar and non-polar interactions, protein-ligand molecular docking studies of papain and cations of both AAILs, have been performed using AutoDock Vina and AutoDock tools 1.5.7. The evaluated results corroborated the data obtained by FTIR, spectroscopic studies and enzymatic activity. From docking, activity and spectroscopic studies, it can be observed that at cholinium cation vehemently prefers polar interaction by forming hydrogen bonding with Arg-64 and Asp-158, however, tetraethylammonium cation longs for the polar interactions by forming van der Waals interactions with Ala-162, Val-133 and Val-207. Moreover, at the higher concentration of AAILs no extraordinary distinction between hydrophilic/hydrophobic natures has been observed and both AAILs denatured the papain at higher concentration irrespective of hydrophilic/hydrophobic nature of cation.

Cloning, Expression, Characterization, and Tissue Distribution of Cystatin C from Silver Carp (Hypophthalmichthys molitrix).

Cystatins are proteins, which inhibit cysteine proteases, such as papain. In this study, the 336-bp cystatin C gene (family II, HmCysC) of silver carp (Hypophthalmichthys molitrix) was cloned and expressed in Escherichia coli BL21 (DE3). HmCysC encodes the mature peptide of cystatin C (HmCystatin C), with 111 amino acids. A typical QXXXG motif was found in HmCystatin C and it formed a cluster with Cyprinus carpio and Danio rerio cystatin C in the phylogenetic tree. Quantitative real-time polymerase chain reaction analysis indicated that HmCysC was transcribed at different levels in five tested tissues of silver carp. Following purification with Ni2+- nitrilotriacetic acid agarose affinity chromatography, HmCystatin C displayed a molecular weight of 20 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified HmCystatin C had strong inhibitory effects toward the proteolytic activity of papain. Immunochemical staining with anti-HmCystatin C antibody showed that HmCystatin C was widely distributed in silver carp tissues. These results collectively demonstrated the properties of HmCystatin C, providing information for further studies of cystatins from fish organisms.

Influence of Bioregulators on Latex Yield and Proteolytic Activity of Papain Extracted from Field-grown TNAU Papaya CO. 8 under the Natural Incidence of Papaya Ring Spot Virus

The cultivation of papaya for both fruits and commercial papain extraction drastically suffers often due to the devastating incidence of papaya ringspot virus disease. The present study aimed to find out whether the papain yield and quality are influenced when bioregulators are applied to manage papaya ring spot virus (PRSV) incidence or as a measure to limit the height of the crop in TNAU Papaya CO.8 variety grown in the open field with the natural incidence of PRSV. Three growth retardants [250 ppm of prohexadione calcium (G2), 500 ppm of mepiquat chloride (G3) and 500 ppm of chlormequat chloride (G4)] and three stress tolerance promoting bioregulators [100 ppm of methyl jasmonate (S2), 100 ppm of salicylic acid (S3) and 0.6 g/ l of strobilurins (S4)] were employed in the study. Water spray (G1 and S1) was kept as control. The treatments were imposed at 4th and 8th months after planting. The growth retardants were initially sprayed and, in the subsequentweek ,the stress tolerance promoting bioregulators were sprayed as per the recommended doses. Among the combinations tried, spraying 250 ppm of prohexadione calcium followed by water spray (G2S1) a week later recorded the lowest plant height (140.79 cm) at the time of fruiting. The treatment combination G 2S2 involving 250 ppm of prohexadione calcium and 100 ppm of methyl jasmonate registered a higher number of fruits (42.17). The combination of 250 ppm of prohexadione calcium along with 100 ppm of salicylic acid (G2S3) was found to be the most effective combination to manage PRSV without compromising papain production and to enhance the proteolytic activity significantly (44378.36 TU g-1) as compared to other combinations. The treatment combination of 500 ppm of mepiquat chloride and 100 ppm of salicylic acid (G3S3) favored higher dry latex yield apart from a significant reduction in PRSV symptoms. Both these treatment combinations (G2S3 and G 3S3) merit due consideration in PRSV management for commercial fruit and papain production in papaya

Utilização de creme de papaína e extrato liofilizado das raízes de salsa (Petroselinum crispum) contra Escherichia coli multirresistente em abscesso em equino

Background: Due to the bacterial resistance to conventional antibiotics, studies on natural products with antibacterial or bactericidal activity are becoming more and more frequent. Among multi-resistant bacteria, Escherichia coli is a producer of extended-spectrum beta-lactamase (ESBL). Papain, coming from papaya latex (Carica papaya), stands out for its capacity to degrade the devitalized tissue that delays the healing process. Parsley (Petroselinum crispum) has been studied, mainly for its diuretic, antioxidant and antimicrobial properties. The aim of this work was to report the use of these two phytotherapic agents in an equine presenting abscess infected by multi-resistant ESBL producing E. coli.Case: A four and a half year old male neutered equine weighing 400 kg of undefined race (SRD) was admitted to the Veterinary Hospital of the Paranaense University (UNIPAR), presenting an increased volume on the left side of the middle third of the neck, one year ago, probably due to an intramuscular injection in the splenius muscle performed by the animal’s owner, who did not remember which drug had been applied. Physical examination revealed a characteristic abscess lesion that involved the subcutaneous and muscular tissue of approximately 10 cm in diameter, and presented a fistulous trajectory with purulent content drainage and pain upon palpation. The animal was initially submitted to surgical drainage of the abscess and to systemic treatment with ceftiofur. In view of the failure of the therapy proposed, the lesion was treated with 2% papain cream and, subsequently, in association with lyophilized parsley extract (Petroselinum crispum) after the identification of the presence of multi-resistant ESBL producing E. coli, isolated from the lesion and identified through standard laboratory tests. The use of 2% papain cream reduced the inflammatory process and fibrous tissue. Lyophilized parsley root extract was effective against infection by multi-resistant bacteria.Discussion: The reduction of purulent secretion and the size of the inflammatory process are due to the proteolytic activity of papain, which decreases bacterial adhesion, and the degree of wound contamination, promoting healing. Several studies have indicated the bacteriostatic and bactericidal activity of papain, but in some trials, this effect was only found in a concentration of 10% for Pseudomonas aeruginosa and Staphylococcus aureus, and no effect for E. coli bacteria. In vitro studies have shown that the essential oil of Petroselinum crispum leaves and stems is more effective than the essential oil of seeds against this bacterium. Reports of isolation and bacterial identification of wounds and abscesses of horses present a large amount of microorganisms, but the presence of multi-resistant E. coli is uncommon. In other countries, the presence of this bacterium in wounds and other regions has been described in this species. In the Netherlands and Germany, the presence of ESBL producing E. coli in horses was detected in clinical samples of pus, stomach, uterus, feces and synovial fluid, presenting a real risk of human contamination by these resistant bacteria. In this sense, the study of antimicrobial agents has great scope, being a crucial point in several sectors of the pharmaceutical field. The phytotherapeutic association, used in this case, represents a possible promising, low-cost alternative for the treatment of contaminated wounds, emphasizing the importance of studies involving alternative treatments for bacterial resistance.

Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level.

Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media

The stability of papain was studied in aqueous–organic mixtures by means of residual proteolytic activity along with various spectroscopic analyses (fluorescence and ATR-FTIR combined with isotopic exchange with D 2O). The investigated systems contained 1 or 10% (v/v) of an aqueous buffered solution (pH 8.0) in acetonitrile (ACN), methanol (MeOH) or dimethyl formamide (DMF). The results evidenced that papain retained almost all its catalytic activity after 24 h of incubation in the presence of ACN, and a more compact conformation of the enzyme was detected. Papain suffered an important loss of enzymatic activity (ca. 80%) after 24 h incubation in MeOH although, no global conformational change and minor secondary structure rearrangements were detected. This observation suggests that somehow the active site region was altered. On the other hand, papain suffered a complete inactivation when exposed to those media containing DMF. Fluorescence analyses revealed that an irreversible conformational change took place after 24 h incubation, and a moderate increase in β-sheet and β-turn structures was the most relevant finding when secondary structure was analyzed. The evidences demonstrated that the organic solvents induce a more rigid and compact structure of papain regardless of the organic:buffer ratio investigated. In turn, these modifications affect the active catalytic site in the particular case of MeOH and DMF. These findings were in agreement with the thermo-stability of the enzyme performed after heating at 353 K in all the studied media, that is the presence of ACN did not substantially affect the secondary structure of papain. Nevertheless, the α-helix domain demonstrated to be less thermally stable than the β-sheet domain, turning into aggregated structures after heating, especially in the presence of MeOH and DMF.

Cloning and Expression of cDNA encoding a Cysteine Protease Inhibitor from clamworm and its possible use in managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae)

A cDNA encoding cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum and ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 KDa deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5alpha harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5alpha and control.

Production of bacteriocin by a novel Bacillus sp. strain RF 140, an intestinal bacterium of Caspian Frisian Roach (Rutilus frisii kutum)

Summary Bacteriocins are proteinaceous antibacterial compounds that exhibit bactericidal activity against species closely related to the producer strain. The aim of this research was to investigate the production of bacteriocin by Bacillus spp. isolated from intestinal bacterial flora of the Caspian Frisian Roach (Rutilus frisii kutum). A bacteriocin produced by the bacterium Bacillus cereus strain RF 140 was identified. The antimicrobial activity started at the exponential growth phase and maximum activity was at the stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Bacteriocin was stable at 80°C, but the activity decreased and was lost when the temperature reached 100 and 121°C, respectively. It was resistant to the proteolytic action of papain, catalase and amylase, but sensitive to proteinase K, pronase E and trypsin. Maximum bacteriocin activity was observed in the pH 4-9. This study indicates the importance of the bacteriocin produced by B. cereus strain RF 140 against food-borne pathogenic microorganisms such as L. monocytogenes and C. perfringens, and presents a potential for use as a biopreservative in food.

Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp.

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.

Purification and Characterization of Extracellular Cysteine Protease Inhibitor from Chlorella sp.

An extracellular cysteine protease inhibitor (ECPI) from Chlorella sp. was purified to homogeneity by DEAE-Cellulose column chromatography and Sephacryl S-300 column chromatography. The molecular mass of the inhibitor was estimated to be 284 kDa by SDS-PAGE and 285 kDa by gel filtration on Sephacryl S-300 column chromatography. ECPI retained 100 % of its original activity even after heating at 100°C for 20 min. It showed an inhibitory activity against the proteolytic activity of papain, ficin, chymopapain, calcium activated protease (calpain) and bromelain, but not against trypsin or α-chymotrypsin. ECPI contained 83.4% carbohydrate residues by weight and inhibited papain at a ratio of 1 : 1. The inactivated inhibitor, treated by sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), was reactivated by keeping it at 10°C and pH 8.5 after elimination of SDS and 2-ME.

Purification and Characterization of Intracellular Cysteine Protease Inhibitor from Chlorella sp..

An intracellular cysteine protease inhibitor (ICPI) from Chlorella sp. was purified to homogeneity judging from polyacrylamide gel electrophoresis. The purified inhibitor had a molecular mass of 410kDa and a pI of 5.3. ICPI retained 100% of the original activity even after heating at 100°C for 5 min. It exhibited an inhibitory activity against the proteolytic activity of papain, ficin, chymopapain, calcium dependent neutral protease and bromelain, but not against cathepsin B, pepsin, trypsin, or α-chymotrypsin. The inhibitory activity was inactivated by α-chymotrypsin. ICPI contained 76% carbohydrate residues by weight and inhibited papain at a ratio of 1:3. From these results, we assume that ICPI is a novel type of cysteine protease inhibitor.

Stability and proteolytic activity of papain in reverse micellar and aqueous media: A kinetic and spectroscopic study

The stability and proteolytic activity of papain were studied in reverse micellar systems, and in aqueous media. In reverse micelles the maximum activity obtained was 80% of the enzyme activity in aqueous solution. Higher papain stability was found in reverse micellar systems compared with that in aqueous solution with half-lives of 24 and 10 days respectively. Electron spin resonance (ESR) spectroscopy studies of aqueous and reverse micellar systems were performed in an attempt to explain the observed enzyme stability and activity profiles. For this purpose a spin label--TEMPOacetamide--was covalently linked to the Cys-25 residue of the papain active center. ESR spectra of labeled papain indicated that catalytic activity of papain could be related to the conformational rigidity near the reaction center. The lower activities obtained in reverse micelles could be a result of the greater degree of mobility and polarity observed in these systems, which can be attributed to papain unfolding. The greater stability found for papain in reverse micelles could be the result of the limited extent of this denaturing process owing to the organized surfactant molecules around the enzyme.

Phospholipid-Papain Interactions

Abstract Interaction of six synthetic phospholipids with papain was studied by differential scanning calorimetry. Papain and phospholipid influenced mutually each other's main transition temperature, the enthalpy of main transition and the half with of the transition suggesting the existence of papain—hospholipid interactions. Multivariate methods proved that the length of apolar fatty acid chains of phospholipids is responsible for the effect, indicating hydrophobic interaction between papain and phospholipids. The efficacy of phospholipids increases with growing length of fatty acid chain, the grade of methylation of polar head group of phospholipids influences to a lesser extent the interaction. The presence of phospholipids has a negligible effect on the proteolytic activity of papain.

Effect of thermal treatments on immunoreactivity and proteolytic activity of papain.

Thermal treatment of papain affects somewhat less its immunoreactivity than its proteolytic activity, especially when higher temperatures and longer heat exposures are applied. Immunochemical methods seem to be, in this respect, more advantageous for papain determination in thermally processed food samples (e.g. pasteurized beer) than assays of proteolytic activity. The proteolytic activity is substantially influenced by the type of substrate used. The following substrates affect the proteolytic activity of partly inactivated papain in an increasing order: low-molecular weight substrate BAPA (N alpha-benzoyl-D, L-arginine-p-nitroanilide) less than HPA (hide powder azure) less than hemoglobin. A protective effect of albumin on papain thermoinactivation was found using both immunochemical and proteolytic activity assays.

Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase

Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, whereas similar treatment of sparse cells causes only a marginal increase in cyclic AMP formation. To determine which membrane protein(s) is altered under conditions which result in proteolytic activation of adenylate cyclase, purified plasma membranes and intact normal rat kidney cells were subjected to limited proteolysis and membrane proteins analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Membranes prepared from intact confluent normal rat kidney cells exposed to mild trypsinization showed a decrease in proteins of 56,000, 46,000, 37,000, and 32,000 daltons. Trypsin treatment of intact, sparse cells does not activate the cyclase system and does not lead to modification of the 46,000-dalton membrane protein. Treatment of purified normal rat kidney plasma membranes results in the loss of numerous bands in the high molecular mass region (>150,000 daltons) as well as decreases membrane proteins of 56,000, 49,000, 46,000, and 23,000 daltons. Compared with trypsin, the proteolytic action of papain appears to be quite specific, causing a discernible decrease in only the 46,000-dalton protein. The correlation between modification of the 46,000-dalton membrane component and the activation of the cyclase system suggests that perhaps this protein is proteolytically modified to elicit activation of adenylate cyclase.

Proteolytic Activity of Papain

Abstract A method based on the ultraviolet absorption of the hydrolytic products of casein digestion and expression of the results as tyrosine equivalents was adopted in 1966 by the Pharmaceutical Manufacturers Association and the Food Chemicals Codex. Based on the collaborative data obtained in 2 studies the method has been adopted by the AOAC as an alternative to 20.114–20.117.

Isolation and determination of non-diffusible sialofucohexosaminoglycans derived from brain glycoproteins and their anatomical distribution in bovine brain.

Abstract— Glycoproteins in brain tissue were assayed by determining the amount of N‐acetylneuraminic acid (NANA), hexosamine, hexose, and fucose present in glycopeptides released by the proteolytic action of papain on the defatted protein residue that remains after treatment of the sample with chloroform‐methanol (2:1 and 1:2, v/v). Diffusible and non‐diffusible glycopeptides (sialofucohexosaminoglycans) were released by proteolysis. The procedure demonstrated that successive treatment of brain tissue with chloroform‐methanol (2:1, v/v) and chloroform‐methanol (1:2, v/v) removed all of the gangliosides present in the tissue.A 1 hr autolysis of rat brain tissue had no effect on the amount of glycopeptides recovered from the tissue. The carbohydrate composition of the non‐diffusible sialofucohexosaminoglycans was also unaffected.Areas of the brain that are enriched in neuronal cell bodies contained a higher concentration of gangliosides and glycoproteins than areas that consist largely of myelinated fibre tracts. On the other hand, there was a greater concentration of glycoprotein relative to that of gangliosides in areas that consist predominately of myelinated fibre tracts and glia than in areas enriched in neuronal cell bodies. The concentration of non‐diffusible sialofucohexosaminoglycans in whole bovine brain was less than that in whole rat brain. The non‐diffusible sialofucohexosaminoglycans from whole bovine brain contained less fucose and NANA per mole of hexosamine and hexose than non‐diffusible sialofucohexosaminoglycans isolated from whole rat brain. The non‐diffusible sialofucohexosaminoglycans isolated from bovine cerebral white matter were lower in fucose and NANA content per mole of hexose and hexosamine than those isolated from other brain areas. It is suggested that the fucose and NANA content of the non‐diffusible sialofucohexosaminoglycans associated with myelinated axons and (or) glia is less than that of the non‐diffusible sialofucohexosaminoglycans associated with the nerve cell body.

Compositional patterns of sialofucohexosaminoglycans derived from rat brain glycoproteins

Glycoproteins in whole rat brain were assayed by determining the carbohydrate content of the glycopeptides released by the proteolytic action of papain on the defatted protein residue which remained after extraction of the tissue with chloroformmethanol (2:1 and 1:2, v/v). Dialyzable and nondialyzable glycopeptides (sialofucohexosaminoglycans) were released by proteolysis. The N-acetylneuraminic acid (NANA) content of the solubilized sialofucohexosaminoglycans and the gangliosides account for 28 and 65%, respectively of the total NANA recovered from the tissue. The remaining NANA is accounted for by free NANA (3%) and NANA that is bound in a papain-resistant fraction (4%). Fractionation of the nondialyzable sialofucohexosaminoglycans by column electrophoresis yielded six fractions which were characterized by an increase in NANA content, a decrease in fucose content, and an increase in electrophoretic mobility as the molecular size of the fraction increased. The six fractions consisted of components which had a molecular size that corresponded to a molecular weight of 9900-13,500 as judged by gel filtration. The sugar components consisted of NANA, fucose, galactose, mannose, glucosamine, and galactosamine. Ninety-five percent of the hexosamine was glucosamine. The hexose/hexosamine ratio for five of the six fractions was 1.34. One of the fractions contained two molecules of hexose per mole of hexosamine. The dialyzable sialofucohexosaminoglycans were separated into three fractions by gel filtration. The three fractions consisted of components which had a molecular size corresponding to a molecular weight of 6000 or less. The smallest component lacked fucose. Dialyzable Sialofucohexosaminoglycans contained the same carbohydrates as the nondialyzable Sialofucohexosaminoglycans, except that a higher proportion of the hexosamine and hexose residues, respectively consisted of galactosamine and mannose.

Chromatography on calcium hydroxylapatite of glycoproteins solubilized by triton X-100 from particulates prepared from rat brain

Aqueous solvents are capable of extracting only 10% of the glycoproteins of the combined mitochondrial and microsomal fractions prepared from rat brain. Solutions that contain 0.5% (v/v) Triton X-100 extract 60% of the glycoproteins. Triton-solubilized glycoproteins can be fractionated by chromatography on calcium hydroxylapatite columns with 100% recovery, although considerable cleavage of the protein-bound N-acetylneuraminic acid occurs during the procedure. Approximately 20% of the applied glycoproteins are not adsorbed to the gel and are recovered with a 2.3-fold purification. A large part of the glycoproteins that adsorb to the gel fail to adsorb upon rechromatography and are recovered with a 2- to 3-fold purification. Glycoproteins present in whole rat brain tissue or tissue extracts were assayed by determining the amount of N-acetylneuraminic acid and hexosamine present in the glycopeptides released by the proteolytic action of papain on the defatted protein residue that remains after extraction of the sample with chloroform-methanol (2:1, v/v). Dialyzable glycopeptides (DGP) and nondialyzable glycopeptides (sialofucohexosaminoglycans, SFHG) are released by proteolysis. The ratio of SFHG/DGP recovered from the glycoproteins present in various extracts and purified fractions prepared from these extracts remains constant, suggesting that both of these glycopeptides are derived from the same glycoprotein or glycoproteins.