Servaas Visser

Phenotyping of bovine milk proteins by reversed-phase high performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major they protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.

Antibacterial and Antiviral Effects of Milk Proteins and Derivatives Thereof

Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral properties of milk proteins and milk protein derivatives. The latter include chemically modified proteins and enzymatically induced peptides. If such peptides are released by enzymes present within the digestive tract (e.g. trypsin or pepsin), it is likely that they play a role in the health defense system. This is especially the case when the active fragments can survive the intestinal conditions long enough to arrive at the right place to exert their beneficial function. In the first part of this paper attention is paid to the antibacterial proteins lactoferrin, lactoperoxidase, and lysozyme. Furthermore, antibacterial peptides originating from caseins and whey proteins are described. The second part reports on studies of antiviral effects of milk proteins and derivatives thereof. Special focus is directed to the antiviral action towards the human immunodeficiency virus (HIV) and the human cytomegalovirus (HCMV). Unmodified milk proteins are generally not active against these viruses. An exception is lactoferrin, which shows significant antiviral activity against both HIV and HCMV. Several other milk proteins tested showed strong antiviral effects only after chemical modification, i.e. by making them polyanionic (for anti-HIV activity) or polycationic (for anti-HCMV activity). In a number of cases, conclusions are drawn concerning possible relationships between antibacterial/antiviral activity and molecular structure of the components described.

Identification of two distinct antibacterial domains within the sequence of bovine alpha(s2)-casein.

Two distinct domains with antibacterial activity were isolated from a peptic hydrolysate of bovine alpha(s2)-casein. The digested alpha(s2)-casein was fractionated by cation-exchange chromatography, after which the peptides in the two active fractions obtained were separated by high-performance liquid chromatography and sequenced by electrospray-ionization tandem mass spectrometry. The major component in each active fraction, f(183-207) and f(164-179), was further purified and the antibacterial activity of these components was tested against several microorganisms. Depending on the target bacterial strain, these peptides exhibited minimum inhibitory concentrations between 8 and 99 microM. Peptide f(183-207) exhibited a consistently higher antibacterial activity than f(164-179), although both peptides showed a comparable hemolytic effect. A method of in situ enzymatic hydrolysis on a cation-exchange membrane to obtain a fraction enriched in the most active antibacterial domain is presented. The antibacterial and hemolytic activities are discussed in relation to the structure and hydrophobicity of the peptides.

Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin. In situ enzymatic hydrolysis on an ion-exchange membrane.

Two ion-exchange chromatographic methods are reported for the rapid isolation of antibacterial peptides from lactoferrin (LF). Using the first method, a pepsin hydrolysate of LF was fractionated by bead-based cation-exchange chromatography. After removal of weakly bound material by washing with ammonia, highly purified lactoferricin-B (LFcin-B) was obtained in a single step by elution with 2 M NaCl. Some other cationic peptides, copurified as minor components, were also characterised by N-terminal sequencing, mass spectrometry and antibacterial activity determination. With the second method, cheese whey was filtered through a cation-exchange membrane, and the selectively bound LF was directly hydrolysed in situ with pepsin. Inactive LF fragments were washed off the membrane with ammonia, and a fraction enriched in LFcin-B was obtained by further elution with 2 M NaCl. The membrane method is more rapid and offers several economic advantages.

Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition or hydrophobicity profile. In contrast with some chemically modified (strongly negative) milk proteins, unmodified alpha(s2)-, beta- and kappa-casein, as well as several negatively and positively charged fragments thereof, did not show significant inhibition of virus replication. In fact, HIV-1 replication was elevated in the presence of beta-casein or amphiphilic fragments thereof. Bovine lactoferrin (bLF), a milk protein of 80 kDa, showed considerable inhibitory activity against HIV-1 with an IC50 of 0.4 microM. Modest inhibition was obtained with lactoferricin, a highly positively charged loop domain of bLF, indicating that other domains within the native bLF protein may also be required for inhibition. bLF blocked HIV-1 variants that use either the CXCR4 or the CCR5 coreceptor. In order to obtain further insight into the mechanism of action of this antiviral protein, we selected a bLF-resistant HIV-1 variant. The bLF-resistance phenotype is mediated by the viral envelope protein, which contains two interesting mutations that have previously been associated with an altered virus-host interaction and a modified receptor-coreceptor interaction. These results demonstrate that bLF targets the HIV-1 entry process.

Reversed-phase high-performance liquid chromatographic separation of bovine κ-casein macropeptide and characterization of isolated fractions

From complex mixtures of non-glycosylated and differently glycosylated caseinomacropeptides (CMP; kappa-casein fragment 106-169; M(r) approximately 7000) various fractions were isolated and further purified by reversed-phase HPLC. The fractions were characterized by mass determination and composition analysis and also used in gel-permeation chromatography and NMR studies to investigate their molecular size behaviour as a function of pH, ionic strength, peptide concentration and degree of glycosylation. No evidence was found for association of any CMP fraction as a function of the experimental conditions applied, which is in contrast with suggestions made in the literature. The increased molecular size (apparent molecular mass approx. 30-45 kDa) is rather explained by a large voluminosity of the molecular species due to internal electrostatic and steric repulsion. Furthermore, the susceptibility of some non-glycosylated and glycosylated CMP fractions to enzymic attack by the Glu-specific endopeptidase from Staphylococcus aureus V8 was studied. Initial rates of proteolysis by this enzyme were independent of the degree of glycosylation. Only in the case of highly glycosylated CMP was further hydrolysis to smaller fragments inhibited. Hydrolytic products were identified by electrospray ionization and fast-atom bombardment mass spectrometry.

Antibacterial and binding characteristics of bovine, ovine and caprine lactoferrins: a comparative study.

A cation-exchange chromatographic membrane was used for the rapid isolation of caprine and ovine lactoferrin (LF) from cheese whey. Caprine LF was isolated by one-step cation-exchange chromatography, but the isolation of ovine LF needed a further reversed-phase HPLC purification step. The antibacterial activity of these LFs, determined towards Micrococcus flavus and Escherichia coli, was compared with that of bovine LF. Of the apo-LFs the caprine protein had the highest activity. In all cases the holo-LFs showed low or negligible activity. The antibacterial properties of cationic peptides obtained by peptic hydrolysis of caprine LF were studied against E. coli and M. flavus, and the activity of these peptides was compared with that of bovine lactoferricin. In addition, the binding characteristics of bovine, ovine and caprine LF to bacterial cells of E. coli were investigated with an enzyme-linked binding assay using horseradish peroxidase-bovine LF as a conjugate. Ovine and bovine LF strongly inhibited the binding of the LF complex to the bacterial surface. The iron-free forms of these LFs showed a greater ability for binding to the bacterial cells than the iron-saturated forms. However, several cationic peptides and caprine LF did not inhibit the binding of the LF conjugate although they exhibited a marked antibacterial effect. The results of binding of the LF complex in the presence of LFs from different species and of membrane-active peptides are discussed in relation to the antibacterial activity of these proteins and peptides.

Action of a cell wall proteinase (PI) from Streptococcus cremoris HP on bovine β-casein

SummaryThe specificity of a cell wall proteinase (PI) from Streptococcus cremoris strain HP in its action on bovine β-casein was determined. To this end an enzymic digest (pH 6.2; 15° C) of β-casein was brought to pH 4.6 and the soluble fraction separated by semi-preparative reversed-phase HPLC. Purified peptides were analyzed by amino acid and end-group analysis. Ten chromatographic components were identified, which together accounted for at least seven cleavage sites all being located in the C-terminal fifty-residue part of β-casein. In five cases it concerned a Gln-X or X-Gln peptide linkage. The specificity of this proteinase from S. cremoris HP shows similarity to that reported for a cell wall proteinase from S. lactis NCDO 763 in its action on β-casein.

The antiviral activity of the milk protein lactoferrin against the human immunodeficiency virus type 1

Milk forms a rich source of biologically interesting components and the protein fraction is known to facilitate many different biological functions. In this manuscript, we review the antiviral properties of the milk protein lactoferrin (LF). In particular, we will describe its antiviral activity against the human immunodeficiency virus type 1 (HIV-1).

Determination of molecular mass distributions of whey protein hydrolysates by high-performance size-exclusion chromatography

Abstract Two high-performance size-exclusion chromatographic columns (Superose-12 HR 10/30) were compared for their use in the determination of the molecular mass (Mr) distribution of whey protein hydrolysates. For calibration 21 reference compounds of known Mr (ranging from 500 to 68 000) were used by applying two procedures for least-squares curve fitting. Based on the calibration graphs constructed, three apparent-Mr regions were arbitrarily assigned: Mr(app) 10 000. Different results for the Mr distributions of the whey protein hydrolysates were obtained with the two columns. This was mainly due to a difference in peak resolution, which was better for the Superdex-75 column in the Mr region relevant for the major whey proteins and their hydrolysates. The results were also dependent on the mode of calibration curve fitting used.