Harold E. Swaisgood

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.

Gelation properties of dispersions containing polymerized and native whey protein isolate

Whey protein polymers (WP-polymers) were prepared by heating whey protein isolate below the critical concentration for gelation at neutral pH and low salt conditions. The effects of WP-polymers and salt types (CaCl2 or NaCl) on rheological properties (large-strain and small-strain analysis), water holding properties, turbidity and microstructure of heat-induced whey protein isolate gels were investigated. Replacement of native whey protein isolate with WP-polymers increased fracture stress, fracture modulus, held water, and the translucency of gels. With both salt types, the addition of WP-polymers changed the gel structure from particulate to fine-stranded. However, the effect of WP-polymers on rheological properties was salt specific. Addition of 20‐100% WP-polymers in the presence of 10 mM CaCl2 caused a continued increase in fracture stress. In contrast, protein dispersions containing 30 mM NaCl did not form self-supporting gels when

Sulfhydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase☆

60% WP-polymers were added. Dispersions containing 200 mM NaCl formed self supporting gels at all levels of WP-polymer addition but fracture stresses for gels containing 20‐100% WP were similar. Dispersions containing 80% WP-polymers and 200 mM NaCl had lower gel points (time and temperature) than dispersions with 80% WP-polymers and 10 mM CaCl2. It appeared that CaCl2 was more effective in increasing gel fracture stress while NaCl was more effective in decreasing gelation time. Different gel properties may be prepared by altering the amount of WP-polymers and salt types. q 2001 Elsevier Science Ltd. All rights reserved.

Surface-bound lactate dehydrogenase: Preparation and study of the effect of matrix microenvironment on kinetic and structural properties

Abstract Xanthine oxidase may be isolated from various mammalian tissues as one of two interconvertible forms, viz., a dehydrogenase (NAD+ dependent, form D) or an oxidase (O2 utilizing, form O). A crude preparation of rat liver xanthine dehydrogenase (form D) was treated with an immobilized preparation of crude bovine sulfhydryl oxidase. Comparison of the rates of conversion of xanthine dehydrogenase to the O form in the presence and absence of the immobilized enzyme indicated that sulfhydryl oxidase catalyzes such conversion. These results were substantiated in a more definitive study in which purified bovine milk xanthine oxidase, which had been converted to the D form by treatment with dithiothreitol, was incubated with purified bovine milk sulfhydryl oxidase. Comparison of measured rates of conversion (in the presence and absence of active sulfhydryl oxidase and in the presence of thermally denatured sulfhydryl oxidase) revealed that sulfhydryl oxidase enzymatically catalyzes the conversion of type D activity to type O activity in xanthine oxidase with the concomitant disappearance of its sulfhydryl groups. It is possible that the presence or absence of sulfhydryl oxidase in a given tissue may be an important factor in determining the form of xanthine-oxidizing activity found in that tissue.

Protein digestibility : in vitro methods of assessment

Abstract Rabbit muscle lactate dehydrogenase ( l -lactate: NAD+ oxidoreductase, EC 1.1.1.27) was covalently bound to porous glass beads using conditions which resulted in attachment of only one of the four subunits of each tetramer. Refolded, covalently bound subunits, prepared by washing with strong denaturants and re-exposure to more nearly physiological conditions, were capable of enzymatic activity and recombination with native subunits in solution. Kinetic properties of a similarly modified, soluble enzyme derivative and several immobilized derivatives were compared. An increase in the apparent Michaelis constant for NADH is suggested to be caused by intrapore diffusion limitation; whereas, the large decrease in the constant for pyruvate may reflect a conformational change induced by the matrix environment. The equilibrium dissociation constant for NAD also exhibit a large decrease compared to reported values for native enzyme suggesting a structural change resulting in more favorable binding.

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A.

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.

Characterization of calcium alginate pore diameter by size-exclusion chromatography using protein standards

Abstract The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10−2 min−1. This provides a t 1 2 of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10−2 min−1 and 7 × 10−3 min−1 for uncatalyzed autoxidation and 4 × 10−2 min−1 and 1.1 × 10−2 min−1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.

Purification and characterization of beta-structural domains of beta-lactoglobulin liberated by limited proteolysis.

Size-exclusion chromatography with 2% Ca alginate beads using proteins of known Stokes radii indicated a pore diameter of 80–100 A when eluents of 30 and 150 mm CaCl2 in 50 mm 2-(N-morpholino)-ethanesulfonic acid and 0.01% NaN3 (pH 6.20) were used. Alginate was shown to exhibit noncovalent interactions with 6 of the 13 proteins employed and these interactions were more pronounced at low Ca2+ concentrations. Three negatively-charged proteins eluted earlier than expected and two divalent cation-binding proteins eluted later than expected. The whey proteins α-lactalbumin and β-lactoglobulin both easily penetrated the alginate pores. No proteins had complete access to the total internal volume of the matrix.

Antimicrobial properties of milkfat globule membrane fractions.

Incubation of β-lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, β-lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel β-sheet structure similar to the native protein but the α-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated ΔGDH20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.