Harold M. Farrell

Proton relaxation rates of water in dilute solutions of β-lactoglobulin determination of cross relaxation and correlation with structural changes by the use of two genetic variants of a self-associating globular protein

Abstract In order to relate resonance relaxation behavior to protein structural states, pulse Fourier transform NMR was employed to obtain water proton longitudinal and transverse relaxation rate ( R 1 and R 1 ϱ = R 2 ) of bovine β-lactoglobulins A and B in buffered solutions. Measurements at concentrations from 5 to 100 mg/ml were made at pH 4.65, 6.2 and 8.0, at 30 and 2°C, to monitor specific structural changes. The parameters characterizing the concentration dependence of the observed R 1 and R 2 were used to derive a number of quantities relating to protein-influenced water, including a hydration parameter h . Changes in h under the different sets of conditions were correlated with (a) the irreversible denaturation of this protein at pH 8.0, 2°C and (b) the dimer ⇄ octamer association at pH 4.65, 2°C. Corresponding correlation times, however, were low, indicating cross relaxation which had not manifested itself as nonexponential relaxation because of the large amount of water present. Differences in the extent of the ⇄ octamer association between genetic variants A and B allowed an evaluation of dynamics and extent of hydration from R 2 alone, assuming the absence of intermolecular interactions. Derived parameters were in agreement with hydrodynamic and X-ray values in the literature. Cross relaxation was likewise evaluated and was found to contribute to R 1 to a large extent. The results show that changes in proton relaxation rates in solutions of a globular protein occuring as genetic variants with different physical properties (such as β-lactoglobulin) can be utilized to detect variations in hydration corresponding to changes in molecular association and conformation, as well as to obtain cross relaxation and structural data.

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.

Calcium-induced associations of the caseins: A thermodynamic linkage approach to precipitation and resolubilization

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wymans thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.

Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cows milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.

Conformational analysis of the hydrophobic peptide αs1-casein(136–196)

Abstract Hydrophobic interactions are important in the self-association of milk proteins, including α s1 -casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide α s1 -casein(136–196). The peptide is aggregated at low concentrations (7 μM and above), as determined by 1 H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1 H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while β-turn structures and some aromatic dichroism are conserved at temperatures as high as 70°C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of β-turns and some side chains in α s1 -casein(136–196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of α s1 -casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a ‘molten globule-like’, heat stable, core structure for α s1 -casein.

Determination of the global secondary structure of proteins by Fourier transform infrared (FTIR) spectroscopy

In the development of new foods or in the control of traditional process, protein functionality plays a paramount role. It has long been theorized that changes in protein structure can alter functionality, but there has been a lack of reliable methodologies for observing protein structural changes in ‘real-world’ food samples. Here, a technique for determination of the global secondary structure of proteins using Fourier transform infrared (FTIR) spectroscopy in H2O instead of D2O is assessed and contrasted with other methodologies for structural determinations. A quantitative procedure is presented for preparing and analysing FTIR spectra of proteins for the determination of their global secondary structural components. As an example, an analysis of the FTIR spectra of egg-white lysozyme is presented and correlated with global secondary structure values calculated from its X-ray crystallographic structure. Other examples of FTIR analyses of food proteins are presented with consideration given to qualitative procedures for more rapid structural analysis of processed samples.

A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

Abstract NADP+:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD+ in place of NADP+ and has an absolute requirement for divalent cations. The apparent Km values for dl -isocitrate, Mn2+, and NADP+ were found to be 8, 6, and 11 μ m , respectively. The Mn2+- d s-isocitrate complex is the most likely substrate for the mammary enzyme with a Km of 3 μ m . The properties of mammary NADP+:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering, sedimentation, and circular dichroism studies on the holo- and apoproteins☆

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 A) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 A3), surface (11,840 ± 120 vs 13,470 ± 140 A), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H2O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.

Isolation and solubilization of casein kinase from Golgi apparatus of bovine mammary gland and posphorylation of peptides

Phosphate incorporation from [gamma-32P]ATP into native and dephosphorylated alpha s1-casein is catalyzed by a casein kinase localized in the Golgi apparatus of lactating bovine mammary gland. Casein kinase from the Golgi is activated with either Mg2+ or Ca2+, and increased specific activity is observed with dephosphorylated casein as the substrate. The casein kinase can be solubilized from Golgi apparatus by the non-ionic detergent, Triton X-100. Gel permeation chromatography on Sepharose CL-4B yields a Stokes radius of 10 nm for the detergent-solubilized casein kinase. Dephosphorylated beta-peptide, the amino-terminal peptide from beta-casein, is a good substrate for the solubilized casein kinase. With dephosphorylated beta-peptide, the maximal velocity is 9.1 and 12.0 nmol/min per mg protein with Mg2+ and Ca2+ activation, respectively. The Michaelis constant for beta-peptide is greater with Ca2+ than with Mg2+ (4.8 mg/ml compared to 0.97 mg/ml). However, the Michaelis constant for ATP is not greatly influenced by these metal ions. The Triton X-100-solubilized Golgi enzyme can also catalyze the phosphorylation of peptides, such as fibrinopeptide A and alpha-melanocyte stimulating hormone.