Gel High-performance Liquid Chromatography Research Articles

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg−1 fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.

Chemical cleavage of mismatch (CCM) to locate base mismatches in heteroduplex DNA

This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.

A Critical Evaluation of Tm(FTIR) Measurements of High-Concentration IgG1 Antibody Formulations as a Formulation Development Tool

Fourier-transform infrared (FTIR) spectroscopy was applied for the determination of protein melting temperature (Tm(FTIR)) and to assess the stability predictability of a 100-mg/mL liquid IgG1 antibody formulation. Tm(FTIR) values of various formulations (different pH, buffers, excipients) were compared to the results of a stability study under accelerated conditions (40 degrees C/75% relative humidity), using size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the detection of soluble aggregates and covalent modifications. The highest Tm(FTIR) was achieved at pH 5.5, and, similarly, SE-HPLC and SDS-PAGE results suggested a pH optimum between 5.5 and 6.0. Transition temperatures were comparable for all tested buffers. However, the decrease in the monomer fraction upon thermal storage was the lowest for citrate buffers. Whereas sugars and polyols resulted in an increase in Tm(FTIR) and enhanced monomer fraction after storage, amino acids showed a destabilization according to SE-HPLC analysis, albeit no change or even an increase in the melting temperature was observed. All examples gave evidence that Tm(FTIR) values did not necessarily correspond to the storage stability at 40 degrees C analyzed by means of SE-HPLC and SDS-PAGE. Tm values, e.g., determined by FTIR, should only be employed as supportive information to the results from both real-time and accelerated stability studies.

Stability of wheat proteins in solution

Gluten from defatted wheat flour was used to study the stability of protein extracts during various steps that are routinely made in protein analyses. These included aqueous extraction and short-term storage, handling of chromatographic fractions, freeze-drying and reconstitution. Protein composition was monitored by size-exclusion high performance liquid chromatography (SE-HPLC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Distilled, deionized and sterilized water were used to prepare the gluten solutions (TFA or HCl solutions at pH 3.0) and to prepare elution solvents. In distilled or deionized water there was a decrease of polymeric protein and a corresponding increase in the monomeric components with time unless initial heating was applied. Protein extracts obtained from gluten and solvents prepared with freshly sterilized water showed no change during storage for 4 days at a temperature of 25 °C. Microbiological assays of the water samples supported the view that microbial contaminants were the source of proteases. After elution from SE-HPLC column with acetonitrile/water followed by freeze-drying and re-dissolution, the SE-HPLC profile of the monomeric proteins was unchanged. However, polymeric proteins aggregated, lost solubility and showed an altered SE-HPLC profile. The effect appeared to be due to the presence of acetonitrile and could be avoided by membrane concentration instead of freeze-drying.

Comparison of rat and human alkaline phosphatase isoenzymes and isoforms using HPLC and electrophoresis

Total activity of alkaline phosphatase (ALP) in serum represents the sum of activities of some isoenzymes and their numerous isoforms derived from various tissues of the organism. The aim of this work was to separate ALP isoenzymes and their isoforms in rat and human serum, compare the properties of serum ALP isoforms in rats and humans, and determine the usefulness of some analytical methods for specific measurements of ALP isoenzyme and isoform activities. Two methods of separation, i.e. high-performance liquid chromatography (HPLC) and agarose gel electrophoresis were chosen. The combination of HPLC with electrophoresis of the eluted fractions and with ALP inhibition methods (urea, l-phenylalanine), inactivation (heat) and precipitation (wheat-germ lectins) enabled the identification of isoenzymes and isoforms of ALP in serum. Using chromatography and a post-column reactor, three isoforms of the intestinal isoenzyme and one bone isoform of a tissue non-specific isoenzyme were detected. Rat serum differs significantly from human as regards activities of intestinal and hepatic isoforms, whereas the properties of the bone isoform are similar in both species. Our HPLC method offers a higher resolution than agarose gel electrophoresis with respect to ALP subfractions in rat serum.

Protein tyrosine nitration in rat brain is associated with raft proteins, flotillin‐1 and α‐tubulin: effect of biological aging

Protein 3-nitrotyrosine (3-NY) immunoreactivity of rat brain homogenate was localized to a ca. 50 kDa protein band by western blot (WB) analysis. The nitrated proteins were localized to the raft fraction obtained by centrifugation of the homogenate in a sucrose density gradient, which contained specific raft markers such as flotillin-1 and caveolin-1. Purification of the nitrated raft proteins either by a combination of reversed-phase high-performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or by immunoprecipitation (IP) with protein- and modification-specific antibodies coupled to WB and HPLC-electrospray ionization-tandem mass spectrometry (ESI--MS/MS) analysis allowed us to identify two proteins modified by 3-NY: flotillin-1 and alpha-tubulin. Both alpha- and beta-tubulin were detected in the rat brain raft fraction as abundant proteins, which co-immunoprecipitate with flotillin-1 and caveolin-1. Importantly, some protein-protein interactions in rafts were disrupted in 3-NY-containing proteins, e.g. caveolin-1 was dissociated from a complex with flotillin-1 and alpha-tubulin. The analysis of age dependencies did not show any significant change in protein nitration and expression of flotillin-1 and alpha-tubulin, but a decrease in the brain caveolin-1 level for old (34 months) versus young (6 months) rats. The putative mechanism of nitric oxide synthase (NOS) activity regulation by the level of caveolin expression and raft protein-protein interactions is discussed.

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.

Distal Substrate Interactions Enhance Plasmepsin Activity

Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb alpha-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase.

Characterization and biochemical analyses of venom from the ectoparasitic waspNasonia vitripennis (Walker) (hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.

Polysaccharides extracted from the leaves of Plantago palmata Hook.f. induce nitric oxide and tumor necrosis factor-α production by interferon-γ-activated macrophages

Ethnopharmacological data from eastern African traditional uses of Plantago palmata leaves suggest that some therapeutical activities could be dependent on their content in polysaccharides (PS). To further investigate immunomodulatory properties of these PS, they were extracted in water from leaves by maceration either at (i) 50 degrees C without filtration (PS50); (ii) 50 degrees C and filtration (PS50F); (iii) 100 degrees C, without filtration (PS100); or 100 degrees C and filtration (PS100F). The extract PS50 was further fractionated by high performance liquid chromatography (gel permeation chromatography) in three fractions namely F1, F2, and F3. The immunomodulatory properties of these four crude extracts and PS50 fractions were investigated by the measurement of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10) production using interferon gamma-(IFN-gamma)-activated macrophages and non-activated macrophages as control. Non-activated J774 cell line macrophages, treated with any of the PS extracts, did produce neither NO, nor TNF-alpha, nor IL-10. In contrast, IFN-gamma-activated J774 macrophages synergized with PS50, but not with the three other crude extracts, to produce both NO and TNF-alpha, but not IL-10. Immunomodulatory effects due to traces of lipopolysaccharides (LPS) in PS extracts and fractions were ruled out by the use of macrophages from C3H/Hej mice known to be very low responders to LPS and similar results were obtained. In addition, F2 fraction from PS50 was particularly active in enhancing NO and TNF-alpha (but not IL-10) production by IFN-gamma-activated C3H/Hej macrophages. These results suggest that PS from P. palmata leaves possess immunomodulatory properties by stimulating NO and TNF-alpha production by activated macrophages.

A β-Glucosidase From &lt;I&gt;Sclerotinia sclerotiorum&lt;/I&gt;: Biochemical Characterization and Use in Oligosaccharide Synthesis

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.

An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions.

The genome of Thermoanaerobacter tengcongensis is estimated to encode 2588 theoretical proteins. In this study, we have vitalized approximately 46% of the theoretical proteome experimentally using a proteomic strategy that combines three different methods, shotgun digestion plus high-performance liquid chromatography (HPLC) with ion-trap tandem mass spectrometry (shotgun-liquid chromatography (LC)/MS), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus HPLC with ion-trap tandem mass spectrometry (one-dimensional electrophoresis (1DE)-LC/MS), and two-dimensional gel electrophoresis plus matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (2DE-MALDI-TOF-MS). Of the 1200 proteins identified, as few as 76 proteins were globally found by all three approaches, and notably, most of these proteins were in the soluble fraction. However, there were a number of unique proteins detected by one method only, suggesting that our strategy provides a means toward obtaining a comprehensive view of protein expression profile. Proteins from the major metabolic pathways are strongly represented on the map, and a number of these enzymes were identified by more than one proteomic method. Based upon the proteins identified in the present study, we are able to broaden the understanding of how T. tengcongensis survives under high temperature environment, whereas several of its properties can not be fully explained by genome data.

Induction of hypoglycaemia in Japanese encephalitis virus infection: the role of T lymphocytes.

We report here development of hypoglycaemia in the convalescent phase of Japanese encephalitis virus (JEV) infection in mice by the induction of antigen-specific Ly1- 2+ T cells in the spleen which mediate hypoglycaemia through the generation of soluble T cell hypoglycaemic factor (TCHF). The TCHF acted in a dose-dependent manner and was found to be trypsin-sensitive and thermolabile. It was purified on Superose-12 high performance liquid chromatography (HPLC) gel filtration column and purified protein migrated as a approximately 25-kD band on SDS-PAGE. The JEV-induced hypoglycaemia coincided with an increased circulating glucagon level, without any alterations in blood insulin and growth hormone concentrations. These effects were mimicked by TCHF. These results indicate that JEV-primed T lymphocytes mediate hypoglycaemia through the production of a soluble hypoglycaemic factor.

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase.

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.

Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins

Salt bridges between self-associating hen egg white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. The self-assembly of bovine insulin was also investigated using cyanogen. Using high-performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). A method was developed incorporating cyanogen, enzymatic digestion, one-dimensional gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein–protein interfaces. The novelty of this approach is the ease with which cyanogen can be administered to a protein sample and the apparent lack of nonspecific cross-linking side reactions interfering with the analysis.

Effects of Microbial Transglutaminase on the Wheat Proteins of Bread and Croissant Dough

ABSTRACT: Transglutaminase is a crosslinking enzyme that is finding increasing use in foods, yet the molecular changes responsible for its effects are not fully understood. Proteins were extracted from bread and croissant doughs that had been treated with transglutaminase and compared to those from control doughs by size exclusion high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Transglutaminase increased the amount of protein extracted in the gliadin fraction in both bread and croissant doughs. In croissant doughs, a corresponding decrease in the protein extracted in the albumin to globulin fraction was seen. In each case, crosslinking of the high molecular weight glutenins was observed. The possible role of each of these changes on the functional properties of baked products is discussed.

α1-Antichymotrypsin Is the Human Plasma Inhibitor of Macrophage Ectoenzymes That Cleave Pro-macrophage Stimulating Protein

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.

Separation of Styrene - Methyl Methacrylate - Acrylonitrile Terpolymers by Composition Using High-Performance Liquid Chromatography

Separation of styrene - methyl methacrylate - acrylonitrile terpolymers by chemical composition was studied using high-performance liquid chromatography (HPLC). Cross-linked polyacrylamide (PAA) gel or polyacrylonitrile (PAN) gel was used as a stationary phase for normal-phase HPLC and cross-linked polystyrene (PS) gel for reversed-phase HPLC. Each system was based on a solvent gradient, and the separation was governed by an adsorption mechanism. Terpolymers were successfully separated by the cross-fractionation, i.e., the combination of two types of HPLC systems with PAN and PAA columns or PAN and PS columns. The chemical composition distribution (CCD) of a high-conversion terpolymer was analyzed by this cross-fractionation technique with the combination of PAA and PAN columns. The CCD obtained experimentally was reasonably consistent with that of theoretical calculation.

Biogenic Amines in Vacuum-Packaged and Carbon Dioxide-Controlled Atmosphere-Packaged Fresh Pork Stored at −1.5°C

Biogenic amines are formed in foods as a result of amino acid decarboxylation catalyzed by bacterial enzymes. When consumed in sufficient quantities, these compounds will cause headache, hypertension, fever, and heart failure. Technologies such as vacuum packaging and carbon dioxide–modified atmosphere packaging (CO2-MAP), when combined with low-temperature storage (−1.5°C), allow fresh pork to have a storage life long enough for export to overseas markets. During low-temperature storage of pork in these packaging systems, the lactic acid bacteria (LAB), which possess the enzymes for biogenic amine formation, dominate the microflora. The objectives of this study were to determine the quantities of biogenic amines in packaged fresh pork, to monitor LAB growth, and to determine the storage life by sensory evaluation. Vacuum-packaged and CO2-MAP pork were stored at −1.5 ± 0.5°C for 9 and 13 weeks, respectively. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine concentrations were determined weekly by high-performance liquid chromatography and capillary gel electrophoresis. LAB and carnobacteria were enumerated weekly. Samples were evaluated for odor and appearance. The CO2-MAP was successful in delaying bacterial growth and the development of unacceptable off-odors compared with the vacuum packaging. The storage lives of the vacuum-packaged and CO2-MAP pork were 5 and 13 weeks, respectively. High-performance liquid chromatography was the superior method for biogenic amine quantification. Tyramine and phenylethylamine in pork of both packaging treatments approached levels considered to be potentially toxic. Given Canada's increasing role in the export of fresh meat to foreign markets, it is recommended that the formation of biogenic amines in vacuum-packaged and CO2-MAP pork be further investigated.

Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin

Abstract Equine whole casein was hydrolysed by chymosin and some peptides generated were characterised by microsequencing after reversed-phase high performance liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis. β-Casein was readily hydrolysed into amino- and carboxy-terminal fragments after cleavage of the Leu190–Tyr191 bond. These two fragments seemed to be resistant to further chymosin hydrolysis on incubation for up to 24 h. Equine κ-casein was purified by affinity chromatography on immobilised wheat germ agglutinin. O-Glycosylated κ-casein was found to represent less than 6.8% of the equine casein components. Equine κ-casein was also hydrolysed and para-κ-casein and glycomacropeptide were generated after cleavage of the Phe97–Ile98 bond.