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Structural properties of whey protein (WP)/gum arabic (GA) coacervates were investigated by measuring the diffusivity of WP and GA in their coacervate phase as a function of pH by means of three different complementary techniques. The combination of these measurements revealed new insights into the structure of coacervates. Nuclear magnetic resonance (NMR) measured the self-diffusion coefficient of the GA in the coacervate phase prepared at various pH values. Fluorescence recovery after photobleaching (FRAP) was measured using a confocal scanning laser microscope. The WP and GA were covalently labeled with two different dyes. The time of fluorescence recovery, related to the inverse of the diffusion coefficient, was evaluated from the measurements, and the diffusivity of the WP and GA on a long time scale could be individually estimated at each pH value. Diffusing wave spectroscopy (DWS) combined with transmission measurement was carried out in the coacervate phase, and the diffusion coefficient, corresponding to the averaged diffusion of all particles that scattered in the system, was calculated as a function of pH. Independently of the technique used, the results showed that the diffusion of the WP and GA within the coacervate phase was reduced as compared to a diluted biopolymer mixture. NMR, DWS, and FRAP measurements gave similar results, indicating that the biopolymers moved the slowest in the coacervate matrix at pH 4.0-4.2. It is assumed that the diffusion of the WP and GA is reduced because of a higher electrostatic interaction between the biopolymers. Furthermore, FRAP results showed that in the coacervate phase WP molecules diffused 10 times faster than GA molecules. This result is very relevant because it shows that WP and GA move independently in the liquid coacervate phase. Finally, DWS measurements revealed that the coacervate phase rearranged with time, as evidenced by a decrease of the diffusion coefficient and a loss of the turbidity of the sample. A more homogeneous transparent coacervate phase was obtained after a few days/weeks. Faster rearrangement was obtained at pH 3.0 and 3.5 than at higher pH values.

The milk proteins β- and κ-casein are in a monomeric state below their critical micelle concentration (cmc) and spontaneously self-associate above the cmc, at or above room temperature. Small-angle neutron scattering (SANS) measurements at 5 g L-1 confirmed the high specific volume of these micelles, and 1H-NMR and calorimetric measurements showed that the polypeptide chains remain open and their side chains largely flexible in the associated state. The experimental values of parameters used to describe both types of micelle were radius of gyration of 8 nm, scattering radius (in 2H2O) of 11 nm, and interaction radius close to 15 nm. The scattering radius is obtained by assuming a homogeneous sphere, and the interaction radius by assuming hard-sphere-like behavior. The micelle size, structure, and interaction radius were independent of concentration. The interparticle structure factor of the micelles was determined using a polydisperse hard-sphere model, which showed that the wave vector position of the pe...

Carrageenans represent one of the major texturising ingredients in the food industry. They are natural ingredients, which are used for decades in food applications. Carrageenan is a generic name for a family of linear, sulfated galactans, obtained by extraction from certain species of red seaweeds (Rhodophyta). Since natural carrageenans are mixtures of different sulfated polysaccharides, their composition differs from batch to batch. Therefore, the quantitative analysis of carrageenan batches is of greatest importance for both ingredient suppliers and food industries to deliver a constant consumer product and to develop new applications based on their unique intrinsic properties. Nowadays NMR spectroscopy is one of the standard tools for the determination of the chemical structure of carrageenan samples. This review gives an overview of NMR-spectroscopy (both 1 H- and 13 C-NMR) as a powerful tool for the qualitative and quantitative analysis of carrageenan samples. In addition to tables containing chemical shift data for both 1 H- and 13 C-spectra, details about sample preparation, selective degradation and fractionation techniques are included.

The coil-to-helix transition and temperature dependence of the viscosity of commercial κ/ι-hybrid carrageenans produced by the red algae Sarcothalia crispata, Mazaella laminarioides, and Chondrus crispus were studied using rheometry and optical rotation. The structure of these κ/ι-hybrid carrageenans was determined by 1H and 13C NMR spectroscopy combined with monosaccharide composition analysis. The coil-to-helix transitions, measured by polarimetry and rheometry, of the κ/ι-hybrid carrageenans are significantly different from those of pure κ- and ι-carrageenan, and from hand-made mixtures thereof. This provides evidence that the κ/ι-hybrid carrageenans are mixed chains, containing both κ- and ι-repeating units. © 2001 Elsevier Science Ltd. Chemicals/CAS: Carrageenan, 9000-07-1; Monosaccharides; Potassium Chloride, 7447-40-7

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.

The antimicrobial membrane-interacting polypeptide nisin is a prominent member of the lantibiotic family, the members of which contain thioether-bridged residues called lanthionines. To gain insight into the complex biosynthesis and the structure/function relationship of lantibiotics, the final intermediate in the biosynthesis of nisin A was studied by nuclear magnetic resonance spectroscopy. In aqueous solution the leader peptide part of this precursor adopts predominantly a random coil structure, as does the synthetic leader peptide itself. The spatial structure of the fully modified nisin part of the precursor is similar to that of nisin in water. The leader peptide part does not interact with the nisin part of the precursor molecule. Thus, these two parts of the precursor do not influence each other’s conformation significantly. The conformation of the precursor was also studied while complexed to micelles of dodecylphosphocholine, mimicking the primary target of the antimicrobial activity of nisin, i.e. the cytoplasmic membrane. The location of the molecule relative to the micelles was investigated by using micelle-inserted spin-labeled 5-doxylstearic acid. It was observed that the N-terminal half of the nisin part of the precursor interacts in a different way with micelles than does the corresponding part of mature nisin, whereas no significant differences were found for the C-terminal half of the nisin part. In this model system the leader peptide is in contact with the micelles. It is concluded that the strongly reduced in vivo activity of the precursor molecule relative to that of nisin is not caused by a difference in the spatial structure of nisin and of the corresponding part of precursor nisin in water or by a shielding of the membrane interaction surface of the nisin part of the precursor by the leader peptide. Probably a different interaction of the N-terminal part of the nisin region with membranes contributes to the low activity by preventing productive insertion. The residues of the leader peptide part just next to the nisin part are likely to contribute most to the low activity of the precursor.

Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4-->Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4-->Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2-->Gly,Pro-2-->Val, Asp-7-->Ala,Lys-9-->Leu,Ser-10-->Ala/Ser-12-->Ala and Val-11-->Asp/Val-13-->Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18-->Leu,Leu-16-->Lys,Asp-15-->Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.

The lantibiotic, nisin, which is known to interact with membranes of certain Gram-positive bacteria, was studied in three model systems which mimic a membrane-like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non-aqueous environments, at 40 degrees C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non-aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N-terminus.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.

Carrageenan is the generic name for a family of linear, sulphated galactans, obtained by extraction from certain species of marine red algae (Rhodophyta) [1]. They are composed of alternating 3-linked β-d-galactopyranose (G-units) and 4-linked α-d-galactopyranose (D-units) or 4-linked 3,6-anhydro-α-d-galactopyranose (DA-units), forming the disaccharide repeating unit of carrageenans (Figure 1). The sulphated galactans are classified according to the presence of the 3,6-anhydro-bridge on the 4linked galactose residue and the position and number of sulphate groups. The most common types of carrageenan are traditionally identified by a Greek prefix indicating the major component of the sample. To describe more complex carrageenan structures a uniform letter code nomenclature has been developed by Knutsen et al. [2]. The three commercially most important carrageenans are called ι (iota)-, κ (kappa)-, and λ (lambda)-carrageenan, the corresponding letter codes are G4S-DA2S, G4S-DA, and G2S-D2S,6S. Two other types, called μ (mu)and ν (nu)-carrageenan, which are the biological precursors of respectively κand ι-carrageenan, are often encountered in commercial carrageenan.

This paper describes the effect of the kappa/iota-ratio on the physical properties of kappa/iota-hybrid carrageenans (synonyms: kappa-2, kappa-2, weak kappa, weak gelling kappa). To this end, a series of kappa/iota-hybrid carrageenans ranging from almost homopolymeric kappa-carrageenan (98 mol-% kappa-units) to almost homopolymeric-carrageenan (99 mol-% iota-units) have been extracted from selected species of marine red algae (Rhodophyta). The kappa/iota-ratio of these kappa/iota-hybrids was determined by NMR spectroscopy. Their rheological properties were determined by small deformation oscillatory rheology. The gel strength (storage modulus, G') of the kappa/iota-hybrids decreases with decreasing kappa-content. On the other hand, the gelation temperature of the kappa-rich kappa/iota-hybrids is independent of their composition. This allows one to control the gel strength independent of the gelation or melting temperature. The conformational order-disorder transition of the kappa/iota-hybrids was studied using optical rotation and high-sensitivity differential scanning calorimetry. High-sensitivity DSC showed that the total transition enthalpy of the kappa/iota-hybrids goes through a minimum at 60 mol-% kappa-units, whereas for the mixture of kappa- and iota-carrageenan, the total transition enthalpy is a linear function of the composition. With respect to the ordering capability, the kappa/iota-hybrid carrageenans seem to behave as random block copolymers with length sequence distributions truncated from the side of the small lengths. Intrinsic thermodynamic properties (e.g., transition temperature and enthalpy) of kappa- and iota-sequences in these copolymers are close to those of their parent homopolymers. The critical sequence length for kappa-sequences is 2-fold of that for iota-sequences.

A new set of 13 C and 1 H NMR chemical shifts of most common carrageenan types is given relative to DSS as the internal standard according to the IUPAC recommendations. Moreover, the chemical shifts of characteristic signals for pyruvate acetal and floridean starch are reported. Additionally, chemical shifts of common internal standards, such as methanol, DMSO and acetone, were measured at different temperatures and pH values.

Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of kappa-casein. During cheese production rennet cleaves kappa-casein into casein macropeptide and para-kappa-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of kappa-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.

Casein micelles in milk are colloidal particles consisting of four different caseins and calcium phosphate, each of which can be exchanged with the serum phase. The distribution of caseins and calcium between the serum and micellar phase is pH and temperature dependent. Furthermore, upon acidification casein micelles lose their colloidal stability and start to aggregate and gel. In this paper, we studied two methods of acid-induced gelation, i.e., 1) acidification of milk at temperatures of 20 to 50 degrees C and 2) decreasing the pH at 20 degrees C to just above the gelation pH and subsequently inducing gelation by increasing the temperature. These two routes are called T-pH and pH-T, respectively. The gelation kinetics and the properties of the final gels obtained are affected by the gelation route applied. The pH-T milks gel at higher pH and lower temperature and the gels formed are stronger and show less susceptibility to syneresis. By using intramicellar cross-linked casein micelles, in which release of serum caseins is prevented, we demonstrated that unheated milk serum caseins play a key role in gelation kinetics and characteristics of the final gels formed. This mechanism is presented in a model and is relevant for optimizing and controlling industrial processes in the dairy industry, such as pasteurization of acidified milk products.

A series of ι-carrageenans containing different amounts of v-carrageenan (0-23 monomer %) have been prepared from neutrally extracted carrageenan of Eucheuma denticulatum. v-Carrageenan is the biochemical precursor of ι-carrageenan. The conformational order-disorder transition and rheological properties of these carrageenans were studied using optical rotation, rheometry, size exclusion chromatography coupled to multiangle laser light scattering, and high-sensitivity differential scanning calorimetry. The helix forming capacity of ι-carrageenan turns out to decrease monotonously with increasing amount of v-units. In contrast, the rheological properties of ι-carrageenan are remarkably enhanced by the presence of a small amount of v-units, yielding a maximum twofold increase in G′ at 3% v-units. It is concluded that the structure-forming capacity of ι-carrageenan, containing a small amount of v-carrageenan, is significantly higher than that of pure ι-carrageenan. This phenomenon is explained in terms of the balance between the helical content and the number of cross-links between chains, taking into consideration the fact that v-units introduce "kinks" in the chain conformation enabling neighboring chains to connect. Increasing amounts of v-units increase the number of cross-links in the network, resulting in increased gel strength. On the other hand, a reduced length of the helical strands weakens the cross-links between the different chains and, consequently, the gel. © 2002 Wiley Periodicals, Inc. Chemicals/CAS: Carrageenan, 9000-07-1; Gels

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.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

The lantibiotic nisin, produced by Lactococcus lactis , is an antimicrobial peptide characterized by the presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (β-methyl)lanthionine rings, which are formed post-translationally. Nisin is widely used in the food industry as a preservative, since it inhibits the growth of unwanted gram-positive bacteria. One of the objectives of our research is to get insight in the complex biosynthesis and regulation of production of nisin. The structure and function of several biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, biosynthesis, secretion and immunity. An exciting finding is that nisin itself, when added to the culture medium, can induce the transcription of its own structural gene. Another goal is to design and produce altered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stability, solubility or activity, recent results on the protein engineering of residues Ile1, Dhb2, AlaS3, Lys12, AbuS13, Met17, Asn20 and Met21 indicate that (i) residue 1 can be replaced without dramatic loss of activity; (ii) the presence of a Thr residue at position 2 significantly lowers the antimicrobial potency, whereas the presence of a Dha residue at position 2 improves activity; (iii) the replacement ofAlaS3 by AbuS leads to a dramatic loss of activity, probably due to a conformational change in the first lanthionine ring; (iv) the integrity and hydrophobicity of ring 3 are important for antimicrobial activity; and (v) the hinge region between rings 3 and 4 is important but not essential for antimicrobial activity.

A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin. Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP. Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin. A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin. Specific acidic residues in the S1 sub site of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the P1 Arg residue of precursor nisin after which cleavage occurs. The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins. Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP. An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues. It may be involved in binding to the cell membrane or to hydrophobic membrane proteins, or it may serve as the recognition and binding region for the modified, hydrophobic C-terminal segment of precursor nisin. Similar predictions for the tertiary structure and substrate binding are made for the highly homologous protein EpiP, the putative leader peptidase for the lantibiotic epidermin from Staphylococcus epidermidis, but EpiP lacks the aromatic patch. Based on these models, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, but also to design lantibiotic leader peptidases with a desired specificity.

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.

The pulse-radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced alpha or beta subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH-values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second-order on-rate constants measured for the binding of oxygen to Hb3+ are 14 +/- 3 mM-1 ms-1 and 56 +/- 9 mM-1 ms-1, respectively. The relative contribution of the faster fraction is about 0.63 +/- 0.08 of the total oxygenation process. A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret-region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on-rate constants.

Studying the effect of KCl on the Bohr effect of human hemoglobin, it appeared that at low Cl− concentration the alkaline Bohr effect is considerably smaller than it is at a Cl− ion concentration near 0.1 M. The data show that at least part of the Bohr effect, that thus far could not be attributed to a particular residue in hemoglobin, is due to interaction of hemoglobin with anions. The effect of KCl on the Bohr effect shows a striking similarity with the effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect. Based on this a mechanism is proposed which satisfactorily explains the observed salt effect.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexo-phosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr protons than ligation with CO. However, in the presence of IHP the observed NO Bohr effect is smaller than the CO Bohr effect. It is Shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146β and Asp 94β upon addition of IHP.

1. We have estimated the binding constant of 2,3-diphosphoglycerate (DPG) to llama and camel hemoglobin at 37°C and pH 6.7, 7.2, and 7.7 from the DPG induced change in the half saturation pressure (P50). The values obtained were compared with the ones derived from similar measurements on human fetal and adult hemoglobin. At pH 7.2 the binding constant of DPG to llama deoxy-hemoglobin is smaller by a factor of 3 in comparison to camel hemoglobin whilst human fetal hemoglobin binds DPG six times less firmly than the human adult pigment. Camel and human adult hemoglobin have about the same affinity for DPG. 2. It is concluded that in llama hemoglobin an amino acid replacement at the DPG binding site explains the reduced phosphate affinity as compared to camel hemoglobin. In human fetal hemoglobin, in addition to an amino acid exchange, there are alterations of the anatomy of the DPG binding site which result in a poorer fit of the phosphate molecule. 3. Our data show that llama hemoglobin has a lower intrinsic oxygen affinity than camel hemoglobin so that despite the reduced DPG binding constant the oxygen affinity of llama and camel blood is very similar at equal phosphate concentration. Possibly, the amino acid exchange at the DPG binding site has compensated for the low oxygen affinity of llama hemoglobin in the course of phylogeny

In this paper we present a procedure for the isolation of the native bovine alpha chain. The method is based on affinity chromatography. The results show that the ligand-binding properties of the bovine alpha chain are almost identical to those of the human alpha chain. The hybrid alphaB2 betaH2 prepared by mixing bovine alpha chains and human beta chains shows ligand binding properties similar to those of human hemoglobin and different from those of bovine hemoglobin.

The pulse radiolysis technique has been used to study the kinetics of the CO binding to partially reduced methemoglobin. Experiments with horse heart metmyoglobin show that this technique gives results which are in good agreement with those obtained by other methods. The kinetics of the CO binding to partially reduced methemoglobin show two phases, whose amplitudes appear to depend on the degree of reduction in such a way that they can be attributed to methemoglobin molecules with one or two reduced heme groups. In the presence of inositol hexaphosphate the rate of CO binding to partially reduced methemoglobin decreases strongly. With inositol hexaphosphate a slight biphasic behavior is observed independent of the degree of reduction.

The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaptiosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the α chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized β chains is slightly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure, (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species.

SummaryThe degradation of bovine β-casein by plasmin was used as a model system to investigate the applicability of an enzyme recycle reactor for the continuous production and isolation of peptide fractions. A simple, ultrafiltration-type membrane reactor was used incorporating a commercially available hollow-fiber module normally employed for dialysis purposes (artificial kidney). The results were compared with those obtained in a batch reactor process, using HPLC to monitor the course of proteolysis.Peptide fragments were isolated and were characterized using amino-acid analysis and N-terminal end-group determination. Sixteen peptide fragments were identified, which together accounted for virtually all the potential cleavage sites in β-casein. In the system studied the N-terminal half of β-casein appeared to be more sensitive to plasmic hydrolysis than the rest of the molecule.

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.

By pulse radiolysis and fast spectrometry the reduction of methemoglobin and metmyoglobin by hydrated electrons has been followed by measuring the spectral changes in the Soret region. The rate of disappearance of the hydrated electrons has also been measured at 650 nm. In the course of the reaction of methemoglobin with hydrated electrons, three different steps could be distinguished. A first step corresponds to the reduction of the heme iron itself, and appears to be bimolecular. The reaction rate constant of this step observed at 435 nm near the isoionic point (pH 7.3) and designated k435, is 5.8 x 1010 m-1 s-1. The same value was found for the rate of disappearance of the hydrated electrons, k650. This was found in spite of the fact that not all the electrons reacting with the hemeproteins react exclusively with the heme groups. This points to the existence of an activated complex between the protein and the hydrated electron. The two reaction constants do not change upon blocking the β(93)SH groups with iodoacetamide. For metmyoglobin at the isoionic point we found that k435 = k650 = 4.3 x 1010 m-1 s-1. Calculations showed that the rate of the reaction of the hydrated electron with the hemeproteins under investigation approaches a diffusion controlled limit. A second step for methemoglobin was found to be monomolecular, with a half-time of 15 ± 2 µs. It could not be detected in metmyoglobin and it disappeared in methemoglobin upon addition of inositol hexaphosphate, indicating that this step is related to a known quaternary structural change, from the R to the T state. This conformational change appears to occur as soon as one heme group in the methemoglobin molecule becomes reduced. A third step, present both in methemoglobin and metmyoglobin, appears also to be monomolecular, with half-time of about 200 µs at pH 7.3. The reaction rate constant for this step in methemoglobin shows a marked pH dependence, suggestive of a titration curve with a pK near 8. For metmyoglobin, the suggested pK is shifted to higher pH values. We conclude that this third step corresponds to the release of heme bound water at low pH, or of OH- at high pH. Oxygen-binding experiments showed that our experimental conditions were such that the Hill parameter n and the oxygen affinity for hemoglobin, reduced by hydrated electrons, remained unaffected.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.

In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.

A method is described for quantifying the proteolytic action of the milk-clotting enzyme chymosin on small and medium-sized peptide substrates by monitoring the decrease of absorbance at 230 nm during cleavage. The method is illustrated by the determination of the kinetic parameters of the specific splitting of a κ-casein-related hexa- and pentadecapeptide by chymosin. The results are in good agreement with those found earlier with the same enzyme/substrate system by using an automated ninhydrin method. Erroneous results were obtained when the kinetic data were derived from one single progress curve. The significance of initial rate measurements for calculating correct kinetic parameters is briefly discussed. The usefulness of single progress curves measured at different initial substrate concentrations for obtaining information about the mechanism of the enzymic reaction is demonstrated.

Binding of 2,3-diphosphoglycerate (DPG) to both deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) is accompanied by an uptake of protons. A study of this proton uptake as a function of n, the mole to mole ratio of DPG and hemoglobin, yielded adsorption isotherms which could be described with one single association constant. It appeared that at pH 6.8 the proton uptake per molecule of DPG bound is larger for HbO2 than for Hb. The data showed that the binding of DPG to HbO2 is functionally significant.

The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-instol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high-affinity systems with binding constants varying between 105 M−1 and 2 × 109 M−1. 31P NMR spectra of P6-instol were recorded in the presence of hemoglobin as a function of the P6-inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the P6-instol NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-insitol to hemogloin. Analysis of the spectra revealed the occurrence of additional binding of P6-insitol to both Hb and HbCO. This binding was also observed in pH-stat experiments performed at low ionic strength. 31P NMR exoeriments carried out with hemoglobin of which the α-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-insitol A model is proposed which accounts for the enhancement of exchange kinetics in these high-affinity systems. In this model a rapid migration is assumed for P6-insitol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three-site exchange problem were calculated.

SummaryThe influence of pH, temperature and Ca depletion on bovine casein micelle suspensions in D2O containing simulated milk ultrafiltrate was studied by 1H-NMR spectroscopy.In the pH range of 5·8–7·5 the spectrum of the micelles showed very little pH dependence, indicating that no changes occurred in the dynamic behaviour of the proteins constituting the micelle.The NMR spectrum of casein micelles was strongly temperature dependent, particularly in the temperature range of 60–98 °C. Increase in temperature resulted in a strong increase in spectral intensity concomitant with changes in the spectral characteristics. In micelle suspensions these changes were reversible, and indicated that at clevated temperatures the rigid structure of the casein micelle started to melt, leading to an increased mobility of appreciable parts of the proteins in the micelle.Ca depletion of the casein micelles by addition of EDTA resulted in an increase in spectral intensity, which arose from the presence of casein components in the serum phase, The spectrum of these serum phase particlcs resembled closely the spectrum of a solution of total casein in simulated milk ultrafiltrate and was quite different from the spectrum of casein micelles.The implications of these results with respect to models of the structure of bovine casein micelles are discussed.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.

The O 2 affinity of β SH chains is lowered by H + , inositol hexaphosphate (IHP), and CO 2 . As the oxygen affinity of β SH monomers ( β 1 SH ) is lower than that of β SH tetramers ( β 4 SH ), it is possible that IHP and CO 2 exert their influence on the O 2 affinity of β SH chains by increasing the dissociation constant of β 4 SH rather than by a direct effect on the molecule. In order to test for this hypothesis we have measured the O 2 affinity of β SH chains as a function of protein concentration at various concentrations of IHP and inorganic phosphates in the absence and presence of CO 2 . From these data association constants for the binding of IHP to β 1 SH and β 4 SH as well as for the equilibrium 4 β 1 SH ⇌ β 4 SH were calculated. We found that IHP and CO 2 influence the oxygen affinity of β 1 SH . It was furthermore established that inorganic phosphate enhances the stability of β 4 SH while IHP favors its dissociation in monomers.

Bohr factors, φ=Δ logPO2/Δ pH, were measured in chicken hemoglobin (Hb) solutions at various levels of O2 saturation in the absence and the presence of inositol hexaphosphate (IHP). pH changes were induced either by changingPCO2at constant base excess near zero (CO2 Bohr factor, {ie135-1}) or by addition of fixed acid or base at constantPCO2(fixed acid Bohr factor, {ie135-2}). In the presence of IHP in a concentration of about 1.2 mol per mol Hb tetramer the two Bohr factors did not differ, the mean value being −0.61 (pH range, 7.1 to 7.6), independent of O2 saturation (SO2) between 10 and 90%. Removal of IHP significantly decreased {ie135-3} to −0.25) pH range, 7.0 to 7.6), independent ofSO2, whereas the reduction in {ie135-4} was much less pronounced. Thus {ie135-5}, which showed a decrease with increasingSO2, exceeded {ie135-6} at all levels ofSO2. The data show that chicken hemoglobin is capable of binding CO2 as oxygen-linked carbamate. But, at about equimolar concentrations of IHP and the tetrameric hemoglobin, oxylabile carbamate formation is abolished. It is suggested that this interaction between CO2 and the organic phosphate compound accounts for the lack of a difference between the two Bohr factors in avian whole blood (cf. Meyer et al., 1978) where inositol pentaphosphate is present at about the same concentration as tetrameric hemoglobin.

Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z.