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This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.

Novel genetic variants for donkey milk lysozyme and beta-lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N49 --> D, Y52 --> S, and S61 --> N, from the previously published sequence. Three novel genetic variants for donkey beta-lactoglobulins were identified. One of them is a type beta-lactoglobulin I with three amino acid exchanges at E36 --> S, S97 --> T, and V150 --> I (beta-lactoglobulin I B, Mr 18,510 Da). The two others are type beta-lactoglobulins II with two amino acid exchanges at C110 --> P and M118--> T (beta-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D96 --> E, C110 --> P, and M118 -->T (beta-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).

Bovine beta-LG was modified by glycation with lactose in a powdered state or in an aqueous solution. An immunological characterization was performed using monoclonal antibodies with defined epitopes. The results showed that the structural changes were confined to the AB loop region of the molecules when glycation was conducted in a restricted water environment and had little consequences on the association state of glycated beta-LG. The protein conformation was much more extensively modified when glycation was performed in an aqueous solution at 60 degrees C, despite a lower glycation extent. These structural changes were located at the dimer interface (AB loop, GH loop, beta-strand I, and alpha-helix). These results allowed us to establish a relationship between the conformational changes and the modification of the association state of the glycated protein (formation of disulfide bridges between the free thiol groups of two monomers), previously described.

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.

In food processing as well as in human nutrition and physiology, the increasing importance of the Maillard reaction has brought about the need for analytical means to detect and characterize the protein-bound Amadori products. In this paper, we describe a highly selective and sensitive method for detecting peptides glycated with lactose (lactolated peptides) from a complex hydrolysate using electrospray ionization tandem mass spectrometry. Protonated molecular ions of lactolated peptides gave a product-ion spectrum, with the dominant mode of decomposition including cleavage of the O-glycosidic bond followed by dehydration steps giving a characteristic neutral loss of 216 Da. Optimization of the ion marker [M + H]+ - 216, identified as a furylium ion, was investigated. It remained dominant regardless of the nature of the glycated peptides, the collision energy used, or the charge state of the parent ion. An approach for detecting lactolated peptides from protein digest was proposed during reverse-phase high-performance liquid chromatography (HPLC)/electrospray mass spectrometry and reverse-phase HPLC/tandem mass spectrometry using neutral loss scanning. This technique detected picomole amounts of lactolated peptides.

Lactose reacts nonenzymatically with beta-lactoglobulin (beta-LG), the major whey protein, under mild heat treatment and the formation of the complex may be monitored by mass spectrometry. Using Reverse Phase HPLC coupled with Electrospray Ionization MS (ESI-MS) we have measured the global extent of glycosylation and examined the distribution of lactose among the beta-LG glycoforms. Identification of lactosylated sites by trypsinolysis and Tandem MS indicate that, although the glycosylation reaction was non-specific and potentially involved all the reactive sites (alpha- and epsilon-amino groups), beta-LG appeared to have at least two populations of lysine with the distinct ability to react with lactose. These results underline the structural heterogeneity of beta-LG glycoforms, with respect to the number of lactose linked per molecule and to the binding sites involved, which could affect the biological function of beta-LG.