George L. Catignani

Antioxidants and prevention of chronic disease.

The generation of reactive oxygen species (ROS) and other free radicals (R•) during metabolism is a necessary and normal process that ideally is compensated for by an elaborate endogenous antioxidant system. However, due to many environmental, lifestyle, and pathological situations, excess radicals can accumulate, resulting in oxidative stress. Oxidative stress has been related to cardiovascular disease, cancer, and other chronic diseases that account for a major portion of deaths today. Antioxidants are compounds that hinder the oxidative processes and thereby delay or prevent oxidative stress. This article examines the process of oxidative stress and the pathways by which it relates to many chronic diseases. We also discuss the role that endogenous and exogenous antioxidants may play in controlling oxidation and review the evidence of their roles in preventing disease.

Tomatoes and cardiovascular health.

Diet is believed to play a complex role in the development of cardiovascular disease, the leading cause of death in the Western world. Tomatoes, the second most produced and consumed vegetable nationwide, are a rich source of lycopene, beta-carotene, folate, potassium, vitamin C, flavonoids, and vitamin E. The processing of tomatoes may significantly affect the bioavailability of these nutrients. Homogenization, heat treatment, and the incorporation of oil in processed tomato products leads to increased lycopene bioavailability, while some of the same processes cause significant loss of other nutrients. Nutrient content is also affected by variety and maturity. Many of these nutrients may function individually, or in concert, to protect lipoproteins and vascular cells from oxidation, the most widely accepted theory for the genesis of atherosclerosis. This hypothesis has been supported by in vitro, limited in vivo, and many epidemiological studies that associate reduced cardiovascular risk with consumption of antioxidant-rich foods. Other cardioprotective functions provided by the nutrients in tomatoes may include the reduction of low-density lipoprotein (LDL) cholesterol, homocysteine, platelet aggregation, and blood pressure. Because tomatoes include several nutrients associated with theoretical or proven effects and are widely consumed year round, they may be considered a valuable component of a cardioprotective diet. Referee: Dr. John Erdman, Director, Division of Nutrition and Food Science, University of Illinois, 455 Bevier Hall, 905 South Goodwin, Urbana, IL 61801

A fluorimetric assay for available lysine in proteins

Abstract A rapid, sensitive fluorimetric assay has been devised for available (nonconjugated) lysine residues of proteins. The ϵ-amino groups of a protein (1- to 40-μg sample) react with o-phthalaldehyde to produce a fluorescent adduct of the protein, whose fluorescence intensity, corrected for the contribution of N-terminal amino groups, is a linear function of lysine content (r = 0.98 for linear regression of 13 protein and peptide standards with lysine content ranging from 0 to 92 mol per 105 g protein). The reaction, which is specific for free amino groups, approaches completion within 1 min at 21°C ( t 1 2 = 6–12 s ) and requires no preliminary heating or hydrolysis of the sample. Triplicate analyses of samples as small as 1 μg gave an accuracy of 10% or better. The method can also be adapted to analysis of unknown protein samples if conditions are used to eliminate from analyses those samples for which the contribution of N-terminal amino groups to the total fluorescence is not insignificant. Consequently, this method by virtue of its simplicity and sensitivity can be used for routine nutritional analyses for available lysine.

An o-phthalaldehyde spectrophotometric assay for proteinases☆

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.

Protein digestibility : in vitro methods of assessment

Publisher Summary This chapter describes those changes in protein structure that lead to change in the digestibility of the protein and discusses several methods for assaying protein digestibility The biological utilization of a protein is primarily dependent on its digestibility by gastric, pancreatic, and intestinal peptidases; its composition, particularly with respect to the essential amino acids; and the absorption or transport of amino acids and di- and tri-peptides into the blood. In vitro methods plays an important role in assaying protein digestibility, because these methods are more rapid, simple, and potentially could be used commercially for monitoring protein quality. Modifications that commonly affect protein digestibility include proteolysis, thermal unfolding, aggregation, carbonyl-amine reactions, racemization, and cross-linking. The digestion of protein in vivo depends on the accessibility and flexibility of the polypeptide chain. The structure of many proteins is destabilized by the acid conditions of the stomach that aids their hydrolysis and further destabilization by pepsin.

Antimicrobial properties of milkfat globule membrane fractions.

Milkfat globule membranes (MFGMs) were prepared from bovine cream according to standard procedures. These membranes and peptide hydrolysates, which were generated by proteolysis with immobilized digestive enzymes, were screened for antibacterial activity against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Typhimurium, Pseudomonas fluorescens, Bacillus cereus, Lactobacillus acidophilus, and Lactobacillus gasseri. Assays were first performed on beef heart infusion (BHI) plates spotted with test protein-peptide fractions and then seeded with lawns of indicator cells to monitor the zone of growth inhibition. Under these experimental conditions, MFGMs were most active against Salmonella Typhimurium and P. fluorescens. However, antibacterial activity was not seen after plating on Luria-Bertani (LB) medium. We determined that the antimicrobial effects observed on BHI plates were due to the generation of H2O2 by xanthine oxidase, a major protein constituent of the MFGMs, as a result of purine catalysis. This substrate is present in BHI but lacking in LB medium. Evaluation of purified xanthine oxidase alone resulted in analogous data trends. The growth of probiotic Lactobacillus strains were affected only marginally when grown on lactobacilli deMan Rogosa Sharpe plates, suggesting the decreased sensitivity of these bacteria to H2O2. In this study, several MFGM hydrolysates exhibited variable antibacterial activity against test food pathogens on agar plates prepared with M9 minimal media, and this variation was not attributable to xanthine oxidase enzymatic activity. The probiotic microorganisms were mostly resilient to these antibacterial fractions. Bovine MFGM fractions may represent an excellent resource material from which to generate native, naturally occurring biodefensive proteins and/or peptides.

Comparison of the properties of trypsin immobilized on 2 Celite derivatives

Trypsin was immobilized on 2 Celite derivatives and the kinetic properties of trypsin immobilized on these derivatives were determined and compared. Celite was derivatized with organosilane to give aminopropyl-Celite (APC) and a portion of this derivative was then succinylated to give succinamidopropyl-Celite (SAPC). Trypsin was covalently immobilized on APC using glutaraldehyde to activate amino groups and on SAPC using water-soluble carbodiimide to activate surface carboxyl groups. Enzyme loadings were 13.9 and 17.8 mg ml-1 of beads on APC and SAPC, respectively. Using p-tosyl-L-arginine methyl ester as substrate, the catalyst specific activity, KMapp and kcat/KMapp were 17.8 U ml-1 of beads, 3.60 and 21.0 mM-1 min-1, respectively, for trypsin-APC as compared with 24.5 U ml-1 of beads, 3.77 and 20.3 mM-1 min-1, respectively, for trypsin-SAPC. With beta-lactoglobulin as substrate, KMapp and kcat/KMapp were 0.36 and 1.62 mM-1 min-1 for trypsin-APC and 0.54 and 1.39 mM-1 min-1 for trypsin-SAPC, respectively. The pH range for optimal activity was much larger for both immobilized forms as compared with the soluble enzyme. The optimal temperature ranges were 40-50 degrees C for trypsin-APC and 50-60 degrees C for trypsin-SAPC. The two methods of immobilization on Celite gave biocataysts with similar kinetic properties but immobilization on SAPC yielded slightly higher loadings and higher specific activities.

Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein☆

A trypsin-streptavidin (TRYPSA) fusion protein was designed and its expression in Escherichia coli was evaluated. The streptavidin gene was PCR modified and cloned into the pET expression vector. The trypsin gene was subsequently inserted into this plasmid, thus generating a colinear fusion of trypsin and streptavidin genes (pTRYPSA). This engineering strategy was verified, and TRYPSA was expressed after IPTG induction using the E. coli strains, BL21(DE3) and BL21(DE3)pLysS. Standard protein fractions of the cell lysate were prepared and trypsin activity was primarily detected in the periplasmic and inclusion body fractions. Immunoblotting showed a single Western-positive band exhibiting a molecular weight of 39,000 Da. A biotinylated porous glass affinity matrix was prepared and selective adsorption resulted in a one-step purification and immobilization of TRYPSA from crude cell lysate. Trypsin activity was verified using a synthetic substrate. This enzyme bioreactor should serve as an excellent prototype for future studies that will examine the effect of limited proteolysis on functional characteristics of milk proteins, including gelling, emulsifying and foaming properties.

Digestibilities of the protein in various foods as determinedin vitro by an immobilized digestive enzyme assay (IDEA)

The digestibility of the protein in various foods or food components was analyzed using an immobilized digestive enzyme assay (IDEA) system. The assay consists of two bioreactors, one containing pepsin and the other containing trypsin, chymotrypsin and intestinal mucosa peptidases. The fraction of the peptide bonds hydrolyzed during an extent of hydrolysis assay was correlated with independentin vivo determinations of the digestibilities. A correlation coefficient of 0.80 was obtained. The derived linear regression equation can be used to predict digestibility. The method is sensitive to structural modification of protein, as for example, those caused by effects of heat treatment.

The estimation of 'available lysine' in human foods by three chemical procedures.

Seventeen test foods were each analyzed by four methods. Total lysine was measured by conventional amino acid analysis. Reactive lysine was measured with either fluorodinitrobenzene, o-phthalaldehyde or a differential dye-binding procedure. The results were then compared with another groups results from rat growth assays of the same samples for availably lysine. A sample of deliberately heat-damaged milk powder gave a rat assay value corresponding to 64% of its total lysine content; other values were all higher and on average 99% for 7 animal products, and 87% for 9 vegetable products. The correlation coefficient between the two sets of values was 0.95. The ‘reactive lysine’ procedures failed to give a better prediction of the rat values.