Increasing Concentrations Of Trypsin Research Articles

An eco-friendly near infrared fluorescence molecularly imprinted sensor based on zeolite imidazolate framework-8 for rapid determination of trace trypsin

This report depicts an environment-friendly molecularly imprinted fluorescence sensor for rapid determination of trypsin based on new near infrared (NIR) fluorescence carbon dots and metal–organic frameworks. The novel NIR fluorescence carbon dots were facilely synthesized from green plants by one-step solvothermal treatment, and the imprinted sensor was prepared by sol–gel under mild condition. The imprinted NIR fluorescence sensor was characterized by Fourier-transform infrared spectrometry, energy disperse spectroscopy, scanning electron microscopy, X-ray diffraction and transmission electron microscopy. The results showed the introduction of zeolite imidazolate framework-8 can improve the fluorescence response sensitivity and response speed. Under optimized conditions, the fluorescence quenching of the imprinted NIR fluorescence sensor was linear relationship with the increasing trypsin concentration in the range of 0.05–9.0 ng/L with a detection limit of 8.8 pg/L. The developed imprinted fluorescence sensor can selectively recognize trypsin in 1.5 min, which was applied to rapidly and sensitively detect trypsin in serum and urine samples successfully with the recoveries of 95.3–104.1% and 97.7–105.2%, respectively. The as-prepared imprinted fluorescence sensor provides a viable and eco-friendly method for rapidly sensitive detection of trace bio-macromolecule in complex samples.

Impact of digestion conditions on phosphoproteomics.

In the past few years, the focus of phosphoproteomics has shifted from merely qualitative to quantitative and targeted studies. Tryptic digestion is a critical step that directly affects quantification and that can be impaired by phosphorylation. Therefore, we systematically characterized the digestion efficiency of 19 nonmodified and phosphorylated model peptides. Whereas we quantified a strong reduction of tryptic cleavage within phosphorylated PKA motifs (R)-R-X-pS/pT and also R-X-X-pT sequences, (R)-R-X-pY sequences were almost unaffected. Structural prediction implied the formation of salt bridges between R/K cleavage sites and phosphoamino acids pS/pT as the main reason for impaired tryptic digestion. We evaluated different conditions to optimize the digestion of such "resistant" phosphopeptides, yielding a substantial improvement of digestion efficiency. We performed a quantitative large-scale phosphoproteomic analysis of human platelets to validate our findings in a complex biological sample. Here, increasing trypsin concentrations up to a trypsin to peptide ratio of 1:10 led to a significant gain (i) in the overall number of phosphorylation sites (up to 9%) and (ii) in the intensities of individual phosphopeptides, thereby improving the sensitivity of phosphopeptide quantification. Still, for certain sequences, the negative impact of phosphorylation on digestion efficiency will further complicate the analysis of phosphorylation stoichiometry.

Factors Affecting Enzymatic Degradation of Microgel-Bound Peptides

Proteolytic degradation and release of microgel-bound peptides was investigated for trypsin, poly(acrylic acid-co-acrylamide) microgels (70-90 μm in diameter), and oppositely charged polylysine, using a method combination of confocal microscopy and micromanipulator-assisted light microscopy. Results show that trypsin-induced release of polylysine increased with increasing trypsin concentration, decreasing microgel charge density and decreasing peptide molecular weight. While the microgel offered good protection against enzymatic degradation at high microgel charge density, it was also observed that the cationic peptide enabled trypsin to bind throughout the peptide-loaded microgels, even when it did not bind to the peptide-void ones. With the exception of highly charged microgels, proteolytic degradation throughout the peptide-loaded microgel resulted in the generation of short and non-adsorbing peptide stretches, giving rise to the concentration and peptide length dependence observed. A simple random scission model was able to qualitatively capture these experimental findings. Collectively, the results demonstrate that microgel charge density, peptide molecular weight, and enzyme concentration greatly influence degradation/release of microgel-bound peptides and need to be considered in the use of microgels, e.g., as carriers for protein and peptide drugs.

Native Protein Proteolysis in an Immobilized Enzyme Reactor as a Function of Temperature

Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis.

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.

Trypsin-induced changes in energy distribution between photosystem I and photosystem II in pea thylakoid membranes

The influence of “mild” trypsin digestion of pea thylakoid membranes on fluorescence emission and excitation spectra was investigated. With increasing trypsin concentration the intensity of fluorescence emission maximum at 735 nm (attributed to PSI) was enhanced and the ratio F 735/ F 685 increased from 1.47 ± 0.12 for control thylakoids up to 3.15 + 0.23 for thylakoids, treated with 8 μg tryp/mg Chl. This increase was more pronounced when the excited light wavelength was 472 nm. In order to clarify which chlorophyll forms are responsible for these changes in the distribution of light energy between both photosystems, an analysis of fluorescence excitation spectra in “blue” and “red” regions was carried out. In the “blue” region an increase of the ratio of intensities 470/436 was observed. In the “red” region an increase of the intensity of the shoulder at 650 nm is observed. Concomitantly, a shift to shorter wavelengths of the excitation maximum at 680 nm was observed. The data presented support the view that the observed increase of photosystem I energy supply involves an increased contribution of Chl b molecules.

Development of a reproduceable in vitro method for assessing the biodegradation of protein microspheres

Protein microspheres have been used for passive targeting of therapeutic agents by chemoembolisation. The overall efficacy of such systems is influenced by the in vivo degradation of the carrier matrix. In this paper we demonstrate a reproduceable method for assessing the degradation behaviour of protein microspheres using laser light scattering apparatus and a model protease trypsin. Degradation has been quantified in terms of the characteristic time dependent effect of the enzyme on the measured volume concentration of the particles. The latent time ( T 1) before the particles start to swell and the time taken to reduce them to fifty percent of their original volume ( T 50) have been measured. Increasing trypsin concentrations up to 0.4% w/v reduce these values for example, the T 50 of bovine serum albumin microspheres reduces from 150 min in 0.1% w/v trypsin to 50 min in 0.4% w/v. Degradation rate is also dependent on the protein matrix used for example casein microspheres provide a T 50 of 150 min whilst transferrin samples provide values of 400 min. This in vitro method will allow comparison of different matrix proteins and investigation of the effects of changes in the methodology of microsphere preparation.

Protease-Induced Formation of the Sperm Acrosomal Filament.

Filament extension during the sperm acrosome reaction in Sicyonia ingentis is triggered by an egg trypsin-like protease whose action can be mimicked using trypsin. Using biotinylated trypsin and either a fluorescently-labeled or colloidal gold-labeled antibody to biotin, trypsin binding was localized to the anterior granule of the sperm which is exposed upon acrosomal exocytosis. The binding was to proteinaceous material at the base of the granule juxtaposed to the inner acrosomal membrane. Other labeled proteins also bound in the same pattern but only in the presence of unlabeled trypsin; non-proteolytic proteins did not induce filament formation. Binding of all proteins tested occurred slowly over a period of about 30 min. A minimum of 30 min of trypsin exposure was required in order to trigger filament formation, and increasing trypsin concentration did not reduce this time requirement. These results indicate that the protease slowly uncovers a binding site for itself (or other proteins), and then its proteolytic activity is again required to induce filament formation. The protease kallikrein appeared to be a more potent inducer than trypsin, while thrombin and clostripain had no apparent inducing activity.

T-kininogen in rat plasma after nephrectomy and other surgical traumas.

Plasma kininogen is known to increase after nephrectomy in the rat, and we studied the type of kininogen responsible for the increase. Total kininogen in plasma was estimated by measuring the kinins released after incubation with an excess of trypsin, and expressed as micrograms bradykinin equivalents per ml of plasma (bk eq/ml). Plasma Kininogen in 24-hournephrectomized rats increased five-fold over the control values (16.4 +/- 0.8 vs 3.0 +/- 0.3 micrograms bk eq/ml; p less than 0.001). However, we found that the increase in total kininogen was not due to nephrectomy per se, but rather to surgery, since similar increases were found after 24 hours in shamnephrectomized rats (15.5 +/- 0.5 micrograms bk eq/ml), and in rats with a catheter implanted into the carotid artery (14.2 +/- 1.7 micrograms bk eq/ml). The increase in kininogen was proportional to the extent of the surgery since simple exposure of the carotid artery increased kininogen to 5.3 +/- 0.5 micrograms bk eq/ml, and sham carotid cannulation to 8.3 +/- 0.4 micrograms bk eq/ml. Anesthesia without surgery did not affect plasma kininogen concentration. To determine whether the adrenals or the pituitary glands are involved in mediating the changes, plasma kininogen was measured after adrenalectomy and hypophysectomy. Total kininogen in plasma of 24 hour-adrenalectomized rats was sevenfold higher than that of intact rats, also higher than the total kininogen in plasma of sham-adrenalectomized rats (21.6 +/- 1.7 vs. 16.5 +/- 1.1 micrograms bk eq/ml; p less than 0.05). The role of the hypophysis was studied by nephrectomizing rats 12 days after they had been hypophysectomized. Total plasma kininogen in the control group of rats was still four-fold higher than in intact rats. Twenty-four hours after nephrectomy, a further two-fold increase was obtained (12.7 +/- 1.6 vs. 25.6 +/- 2.0 micrograms bk eq/ml; p less than 0.001). Kinins released by trypsin from plasma of the various groups of rats were separated by reserve-phase (C18) high pressure liquid chromatography (HPLC), and then quantitated. Only T-kinin was found to be increased. Unknown kinins, which were partially converted to T-kinin by increasing trypsin concentration from 10 to 80 mg/ml plasma, were also found by HPLC. The results indicate that surgical trauma induces a marked increase in the concentration of T-kininogen in rat plasma. Neither the hypophysis, nor the adrenals seem to be involved in mediating the increase in T-kininogen.

Phosphorylation of the rat hepatic polymeric IgA receptor.

In vivo labeling with [35S]cysteine has identified three transmembrane forms of the rat hepatic polymeric IgA receptor: (i) a 105-kDa core glycosylated precursor; (ii) a terminally glycosylated 116-kDa intermediate; and (iii) a mature 120-kDa form. In the current study we show that the 120-kDa form is phosphorylated. After in vivo labeling with [32P]orthophosphate, all receptor forms were immunoprecipitated from hepatic total microsomes (TM) (with an antireceptor antiserum), separated by NaDodSO4/PAGE, and detected by autoradiography. The 120-kDa form was selectively phosphorylated, whereas the 116- and 105-kDa forms incorporated no detectable 32P. To determine the topology of the phosphorylation sites, hepatic TM isolated from rats labeled in vivo with either [35S]cysteine or [32P]orthophosphate were treated with trypsin. TM were solubilized and receptors were immunoprecipitated from lysates. With increasing trypsin concentrations, the [35S]cysteine-labeled receptor triplet was degraded to a trypsin-resistant doublet of approximately 95 and 85 kDa, indicating that approximately 20 kDa was removed from the receptor endodomain by trypsin. The same treatment removed all detectable 32P from labeled receptors. Furthermore, no 32P was detected in the 80-kDa biliary form of the receptor. Serine was identified as the only phosphorylated residue in acid hydrolysates of 32P-labeled immunoprecipitated receptor. These findings indicate that (i) the 120-kDa form is the only phosphorylated species of the receptor; and (ii) the phosphorylated residues are serine(s) located in the endodomain of the protein.

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core.

Limited proteolysis has been used to probe the subunit structure (Mr = 52,000) of the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Digestion of the complex at 0 degrees C with a low concentration of trypsin produces an inner E2 core that retains the activity for the transacylation reaction and is completely dissociated from the decarboxylase (E1) component. The trypsinized E2 maintains the highly assembled structure and migrates faster than the native E2 in the Sepharose 4B column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the inner E2 core consists of two lipoate-free tryptic fragments, i.e. fragment A and fragment B with Mr = 26,000 and 22,000, respectively. Both fragments apparently fail to bind the E1 component. Fragment A is converted into fragment B by increasing trypsin concentrations. Fragment B is a stable limit polypeptide containing the intersubunit-binding sites for E2. The assemblage of fragment B confers the cubelike appearance of the inner E2 core in electron micrographs. Activity measurements indicate that the larger fragment A, but not fragment B, possesses transacylation activity. It is likely that a critical portion of the active site is present in the 4,000-dalton fragment that is lost during the conversion of fragment A to B.

Effect of proteolysis on the activity of the sodium channel in isolated lobster nerve membrane.

The effect of enzymatic proteolysis on structural and functional properties of the isolated lobster nerve membrane was investigated. The membranes were treated with different amounts of either trypsin or unspecific protease. Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in proteoliposomes prepared with nerve membrane and soybean lipids. The changes in the membrane proteins were followed by electrophoresis in polyacrylamide gradient gels. From the densitometric scan of the gels the relative area for each protein was obtained, and the ratio of enzyme-treated to control areas was evaluated. Under a similar degree of proteolysis catalyzed either by trypsin or by unspecific protease, the sensitive sodium influx is not affected by trypsin, whereas it is about 60% diminished by the unspecific protease. In this condition the zones corresponding to molecular weights of 240,000 and 166,000 daltons appear modified in the electrophoretic gels by both enzymes. The 117,000-dalton range is modified only by the unspecific protease. Increasing trypsin concentration diminishes sodium influx about 60%; and the 240,000-, 166,000-, and 117,000-dalton zones appear modified. A further increase of the protease concentration totally abolishes the sensitive sodium influx and modifies practically all of the membrane proteins. The present results indicate the rather high sensitivity of the membrane sodium channel activity to proteolytic action, and show that the membrane sites that respond to veratridine appear to be highly affected by proteolysis. In contrast, the tetrodotoxin receptor retains its binding capacity even after treatment of the membrane with protease concentrations 1,000 times higher than those affecting the sensitive sodium influx [Benzer and Raftery, 1972; Villegas et al, 1973].

In vivo and in vitro effects of proteases upon adenylate cyclase activity of liver cells and plasma membranes.

1. 1. Several hours after an intraperitoneal injection of papain (3 mg) the marked stimulation of adenylate cyclase activity was observed as well as the significant increase in cyclic AMP content of rat livers. The fluoride-sensitive adenylate cyclase activity was stimulated more than the basal activity. Trypsin, which is not mitogenic, was much less effective on both events, as compared to papain which is mitogenic to the liver in vivo. 2. 2. Papain stimulated the basal adenylate cyclase activity of isolated liver cells in vitro at concentrations larger than 2.5 μg papain per ml, but the sensitivity of adenylate cyclase activity to fluoride was completely lost upon increasing the papain concentration. Trypsin, on the other hand, gave no effect on the basal adenylate cyclase activity of isolated liver cells at enzyme concentrations from 0 to 500 μg/ml. The fluoride sensitivity was lost upon increasing trypsin concentration similarly to the case of papain. 3. 3. Adenylate cyclase activity (both basal and fluoride-sensitive) of isolated liver plasma membranes was stimulated by papain (but not by trypsin) at concentrations as low as about 0.25 μg papain/ml, similarly to the in vivo effect of proteases. Upon increasing the protease (papain, trypsin) concentration further, both the basal and fluoride-sensitive activities of plasma membranes were markedly reduced. 4. 4. Acid mucopolysaccharides of liver plasma membranes were released almost completely by treatment with papain at concentrations larger than 25 μg/ml, whereas trypsin was less effective in degrading proteoglycans associated with plasma membranes. 5. 5. The results were discussed in relation to the possible involvement of protease in mitogenesis of liver cells through the alteration of cell surface architecture and function.

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites.

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.

Comparative studies on the tryptic digestion of actomyosins and myosins from several fish species. II. Changes in Ca2+ and EDTA-ATPases.

Ca2+- and EDTA-ATPase of actomyosins from several fish species and rabbit were assayed during digestion with trypsin. In typical experiments, the initial activation as well as the inactivation with increasing trypsin concentration were confined to the shorter durations. During digestion, Ca2+- ATPase of heat denatured samples of bigeye tuna and carp actomyosins showed patterns which were identical with those of their respective fresh preparations. A higher initial activation was a general feature of the tuna actomyosins, if the assay of Ca2+- ATPase was performed in 60mM KCl. The initial activation of tilapia actomyosin Ca2+- ATPase was of a lesser extent, whereas it was absent from carp, yellowtail and flatfish actomyosins. At the higher KCl concentration (500mM) in the ATPase assay, the response of Ca2+- ATPase was altogether monophasic i.e. devoid of any initial activation even in the case of rabbit actomyosin. EDTA-ATPase pattern of actomyosin digestion by trypsin closely resembled the pattern of Ca2+- ATPase at the higher KCl concentration. The activation of myosin Ca2+- ATPase of tuna species occurred to a lesser extent than that of actomyosin ATPase, and the reversal of the order of stability observed in case of actomyosin ATPase of tilapia and skipjack tuna was no longer noted. In the light of published work, the reasons for these discrepancies have been discussed.