Infrared laser ablation and capture of enzymes with conserved activity.

Infrared (IR) laser ablation at 3 µm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ±â€¯8% at laser fluences between 10 and 30 kJ/m2. Trypsin retained 37 ±â€¯2% of its activity and catalase retained 50 ±â€¯7%. The activity of catalase from tissue was tested using three consecutive 50 µm thick rat brain sections. Two 4 mm2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue.

Cutting edge proteomics: benchmarking of six commercial trypsins.

Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC-MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence specificity. The analysis of autolysis products led to the identification of a number of contaminating proteins and the generation of a list of peptide species that will be present in tryptic digests. Intriguingly, many of the autolysis products were nontryptic peptides, specifically peptides generated by C-terminal cleavage at asparagine residues. Both porcine and bovine trypsins were demonstrated to be tyrosine O-sulfated. Using both a label-free and a tandem mass tag (TMT) labeling approach, a comparison of the digestion of a standard protein mixture using the six trypsins demonstrated that, apart from the least expensive bovine trypsin, the trypsins were equally specific. The semitryptic activity led to a better sequence coverage for abundant substrates at the expense of low-abundance species. The label-free analysis was shown to be more sensitive to unique features from the individual digests that were lost in the TMT-multiplexing study.

A study of the effects of column porosity on gradient separations of proteins.

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.

Effect on performance and carcass characteristics of nursery to finisher pigs fed reduced crude protein, amino acid-supplemented diets.

An experiment was conducted to evaluate the effect of feeding reduced CP, amino acid (AA)-supplemented diets to pigs from weaning to slaughter weight on growth performance and carcass characteristics. Pigs were fed a 19%-16%-14% CP starter-grower-finisher high-CP sequence of diets, respectively, or a sequence of diets reduced in CP by 4 percentage units (3 percentage units in the finisher period) with or without lysine (LYS), tryptophan (TRP), and threonine (THR) supplementation. Pigs fed the low-CP diets without AA supplementation grew more slowly, were less efficient in feed conversion, and developed carcasses that contained a smaller longissimus muscle, greater, average backfat depths, and a lower percentage of muscle compared with pigs fed the high-CP sequence of diets (P < .01). The reduction in growth performance, feed efficiency, longissimus muscle area, and percentage of muscle in pigs fed the low-CP diets was alleviated by LYS, TRP, and THR supplementation (P > .10). Although pigs fed the low-CP diets supplemented with AA had reduced average and 10th rib backfat depths compared with pigs fed the unsupplemented, low-CP diets (P < .01), these fat depth measures remained greater (P < .05) than those of pigs fed the high-CP diets. Feeding reduced dietary CP, regardless of AA supplementation, resulted in reduced heart (P < .10) and liver weights (P < .01) compared with feeding the high-CP diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of primary cultures of pulp cells from bovine permanent incisors.

A primary culture method was established by comparing the different effects of four methods of enzymatic separation--trypsin, collagenase with and without trypsin pretreatment, and a trypsin-collagenase mixture--and five media: DMEM, DMEM and Ham's F 12 mixture, F 12, RPMI 1640 and Medium 199. The trypsin pretreatment/collagenase method was most preferable considering the high number of isolated cells, satisfactory adhesion, good growth and a single population at subconfluence. DMEM and the DMEM/F-12 mixture resulted in the best adhesion, cell growth and cell number at confluence. Primary cells separated by the trypsin pretreatment/collagenase method and cultured in DMEM were responsive to parathyroid hormone at the proliferating stage and had higher alkaline phosphatase activity than cells cultured from gingiva and mucosa after reaching confluence. The long-term cultured cells formed nodules that were slightly mineralized. These results indicate that the cultured pulp cells had properties characteristic of pulp cells in vivo. This enzymatic separation method may be useful in studies of the regulation of pulp metabolism and odontoblast differentiation.

[In vitro studies of pancreatic enzyme substitution]. / In-vitro-Untersuchungen zur Pankreasenzymsubstitution.

In-vitro activity of 14 commercial pancreatin preparations, commonly used in the Federal Republic of Germany, were tested. All had been declared by their manufacturers to contain more than 6000 FIP (Fédération International Pharmaceutique) units of lipase and to be acid resistant. The declared lipase and amylase amounts were found to be present in 11 of the 14 preparations. Three of the 14 preparations, said to be acid resistant were found not to be so in buffer with falling pH values between 4.0 and 2.5, so that there occurred an, at times marked, loss of enzyme activity. Most noticeable was the poor solubility of most preparations at pH 6.6. Only three of the 14 liberated their total enzyme content within 60 minutes, as they should for theoretical reasons, based on the relatively short duodeno-cecal transit time.

Non-competitive enzyme immunoassay of human trypsin 1.

A sandwich enzyme immunoassay was developed for human pancreatic trypsin 1 using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labeled IgG as the second antibody. The entire assay takes 6 h and the detection limit is 0.5 microgram/l. The assay can be performed on sera samples or on discs carrying dried blood spots. Good agreement was found with a radioimmunoassay kit. This simple assay could be widely applied to confirm the elevated immunoreactive serum trypsin described in newborn children with cystic fibrosis.

Heterogeneity among preparations of crude trypsin used to disaggregate the human tonsil.

Crude trypsin is the agent of choice for the disaggregation of human tonsil. A great variability which is not related to specific activity exists among lots of crude trypsin. An efficient lot of crude trypsin is necessary to obtain optimal proportions of tonsillar cells for the purification of lymphocytes and plasma cells by a previously reported technique.

Factors influencing the activity of sterile filtered and heat-sterilized trypsin solutions.

The mode of sterilization (filtration or heat) was found to significantly affect the activity of trypsin solutions. Trypsin activity was substantially reduced in the initial fractions of filtrate passed through asbestos filter pads; heat-sterilized trypsin was satisfactory for transfer of cell cultures grown on glass. Heat-sterilized trypsin may be useful when elimination of filterable organisms is required.