Peter Sellers

Thermal denaturation of β-lactoglobulin: effect of protein concentration at pH 6.75 and 8.05

Previous work on the thermal denaturation of beta-lactoglobulin at about neutral pH and concentrations generally above 50 mg/ml has shown that the temperature of the maximum in the thermogram increases only slightly with concentration. Likewise, there is little if any concentration dependence at acid pH over a wide concentration range. However, so far as we are aware, no work has been described on the thermal denaturation of beta-lactoglobulin in the physiological range of protein concentration and pH appropriate to milk. We report measurements at pH 6.75 and 8.05 in the concentration range 2-120 mg/ml and show that below about 50 mg/ml the position of the maximum becomes strongly dependent on concentration, passing through a minimum near 25 mg/ml and increasing towards the lowest concentrations where measurements were practicable. Moreover, the narrow, well defined and nearly symmetrical thermal transition observed at high protein concentrations contrasts with a broader and more asymmetric curve at lower concentrations. An explanation for the behaviour seen at the lower protein concentrations is suggested, based on the temperature- and concentration-dependent dissociation of the beta-lactoglobulin dimer and an associated conformational transition. The position of the maximum in the thermogram has a marked dependence on the rate of heating down to the lowest rate investigated of 10 degrees C per hour, showing the importance of slow kinetic effects in the denaturation of this protein.

Enthalpies of vaporization of some 1-substituted n-alkanes

Abstract Enthalpies of vaporization at 298.15 K have been determined calorimetrically for some 1-substituted straight-chain alkanes. The results are: Compound ΔH v o /kJ mol −1 Compound ΔH v o /kJ mol −1 1-octene 40.27 ± 0.20 1-bromooctane 55.77 ± 0.25 1-decene 50.43 ± 0.20 1-bromododecane 74.77 ± 0.38 1-dodecene 60.78 ± 0.20 1-bromohexadecane 94.4 ± 1.5 1-hexadecene 80.25 ± 0.42 methyl pentanoate 43.10 ± 0.06 1-hexanol 61.85 ± 0.20 methyl hexanoate 48.04 ± 0.12 1-heptanol 66.81 ± 0.20 methyl heptanoate 51.62 ± 0.48 1-octanol 70.98 ± 0.42 methyl octanoate 56.41 ± 0.50 1-nonanol 76.86 ± 0.75 methyl nonanoate 61.99 ± 0.41 1-decanol 81.50 ± 0.75 methyl decanoate 66.75 ± 0.57 1-dodecanol 91.96 ± 0.59 methyl undecanoate 71.37 ± 0.30 1-tetradecanol 102.2 ± 2.3 methyl dodecanoate 77.17 ± 0.56 1-mercaptodecane 65.48 ± 0.54 methyl tridecanoate 82.68 ± 0.84 methyl tetradecanoate 86.98 ± 0.94 1-chloropropane 28.50 ± 0.21 methyl pentadecanoate 93.49 ± 0.94 1-chlorododecane 71.93 ± 0.32 1-chlorohexadecane 91.8 ± 1.1 An analysis of available data shows that the enthalpy of vaporization for members within seven homologous series, XC m H 2m + 1 , can be expressed as ΔH v o (X, m ) = A x + B x m .

Binding of Sodium Dodecyl Sulphate and Dodecyl Trimethyl Ammonium Chloride to β-Lactoglobulin: A Calorimetric Study

Abstract Thermally induced unfolding of β -lactoglobulin in the presence of surfactants was studied by differential scanning calorimetry (DSC). Anionic surfactants, sodium dodecyl sulphate (SDS), and cationics, dodecyl trimethyl ammonium chloride (DOTAC), were used. In a solution containing a 1:1 molar ratio of SDS and β -lactoglobulin, the protein was stabilised as evidenced by a substantial increase in the unfolding temperature. Further increase of the SDS concentration causes unfolding of the protein. As opposed to these effects of the anionic surfactant, a slight decrease in the unfolding temperature was observed in the presence of DOTAC under similar conditions. An increase of the DOTAC/ β -lactoglobulin molar ratios above 1:1 causes precipitation of the protein. The cationic surfactant could be fairly easily removed from a mixed β -lactoglobulin/DOTAC solution by dialysis. The anionic surfactant appeared to interact more strongly with the protein and the 1:1 molar interaction with SDS was impossible to separate by dialysis. The experimental findings are discussed in terms of possible binding sites for the surfactant and connected to micelle formation of the surfactants which is related in a predictable scheme to temperature and ionic strength effects.

Dissection of Calbindin D9k into two Ca2+-binding subdomains by a combination of mutagenesis and chemical cleavage

Calbindin D9k is a 75‐residue globular protein made up of two Ca2+‐binding subdomains of the EF‐hand type. In order to examine the subdomains independently, a method was devised to selectively cleave the loop between them. Using site‐directed mutagenesis, a unique methionine was substituted for Pro43 in the loop, thus allowing cleavage using cyanogen bromide. Agarose gel electrophoresis shows that the fragments have a high affinity for one another, although less so in the absence of calcium.1H‐NMR spectra of the fragments indicate that the structures of the heterodimers are changed little from that of the intact protein. However, the Ca2+ binding constants of the individual subdomains are several orders of magnitude lower than for the corresponding sites in the uncleaved protein.

Comparison of the Effect of Heating on the Thermal Denaturation of Nine Different β-Lactoglobulin Preparations of Genetic Variants A, B or A/B, as Measured by Microcalorimetry

Three samples each of the pure A and B genetic variants together with three samples of the mixed A/B variants of β-lactoglobulin were studied by differential scanning microcalorimetry. All the samples were characterised with respect to whey protein composition and shown to be almost pure β-lactoglobulin. Other analyses showed only minor amounts of other contaminants but variable degrees of lactolation ranging from <1.9 to 10.4 mol%. Thermograms were recorded at about 3 mg β-lg ml -1 in a 10 mM P i buffer pH 7, with the ionic strength of milk, using a scan rate of 60°C h -1 on both Microcal MC-2 and MCS calorimeters and reasonable agreement was found for the denaturation temperature measured on the two instruments. The biggest source of variation in the thermograms of samples was due to the genetic variant. For the single variants, other differences were in the same order as the variability seen in repeat preparations of the same sample. A slightly greater variation was found among the A/B samples where the one with the lowest degree of lactolation was most stable. However, dialysis of the sample against the P i buffer almost completely eliminated the difference. Qualitatively, all thermograms appear to be the sum of 4 processes centred at temperatures of approximately 55, 77, 105 and 125°C. Compared to the A variant the thermogram of the B variant shows a less prominent shoulder at about 55°C. Also, the broad feature at about 105°C is reduced and the peak at 125°C much enhanced.

Enthalpies of formation of some aliphatic branched ketones

Abstract Enthalpies of combustion and vaporization at 25.0 °C have been measured for some aliphatic branched ketones. Enthalpies of formation at 25.0 °C have been derived for the compounds in the liquid and gaseous states. The results are: ΔH f o (l)/kJ mol −1 ΔH f o (g)/kJ mol −1 2-Methyl-3-pentanone −325.9 ± 0.9 −286.1 ± 0.9 2,2-Dimethyl-3-pentanone −356.1 ± 1.4 −313.8 ± 1.4 2,4-Dimethyl-3-pentanone −352.9 ± 1.1 −311.3 ± 1.1 2,2,4-Trimethyl-3-pentanone −381.6 ± 1.2 −338.3 ± 1.2 2,2,4,4-Tetramethyl-3-pentanone −391.2 ± 1.2 −345.8 ± 1.2 2,6-Dimethyl-4-heptanone −408.6 ± 1.2 −357.7 ± 1.2 2,2,5,5-Tetramethyl-3-hexanone −442.7 ± 2.3 −393.9 ± 2.3 The results are discussed in terms of steric effects introduced by methyl substitution in the α- and β-positions. An Allen-type bond energy scheme is applied to the results and the resulting equation used to derive a value of approximately 20 kJ mol−1 for the steric energy in 2,2,4,4-tetramethyl-3-pentanone.

Enthalpies of combustion and formation of 2-nonanone and 2-dodecanone

Abstract Enthalpies of combustion and vaporization at 298.15 K have been measured for 2-nonanone and 2-dodecanone. Enthalpies of formation at 298.15 K have been derived for the compounds in the liquid and gaseous states. The results are: ΔH f o (l)/kJ mol −1 ΔH f o (g)/kJ mol −1 2-nonanone −397.21 ± 1.74 −340.78 ± 1.74 2-dodecanone −476.09 ± 2.37 −404.26 ± 2.45

Thermodynamics of Ca2+ binding to calmodulin and its tryptic fragments

The binding of Ca2+ to calmodulin and its two tryptic fragments has been studied using microcalorimetry. The binding process is accompanied by the uptake or release of protons, depending on the ionic strength. With no added salt, the total enthalpy change for the binding of four calcium ions to calmodulin is -41 kJ mol-1 but in the presence of 0.15 mM KCl delta Htot is +17 kJ mol-1. The mode of binding of Ca2+ is also completely different with and without added salt. It is also shown that for the C-terminal fragment of calmodulin, TR2C, the drastic reduction in delta Gtot for the binding process on increasing the ionic strength is largely an enthalpic effect. Domain interactions in calmodulin are indicated by the fact that the sum of the enthalpies of calcium binding to the two tryptic fragments is not the same as the total binding enthalpy to calmodulin itself. The binding of Ca2+ to calmodulin has also been studied calorimetrically at different temperatures in the range 21-37 degrees C. delta Cp is large and negative in this interval.

Enzyme kinetics. Thermodynamic constraints on assignment of rate coefficients to kinetic models.

Enzyme-catalyzed reactions are complex in that they proceed from an initial set of reactants to a final set of products via a mechanism comprising more than one elementary reaction. This is because catalysis not only involves the binding and dissociation of reactantls and productls (terminal species) of the overall catalyzed reaction to and from the enzyme, but also transitions between enzyme states. In general, this does not involve the net consumption of enzyme because it is “cycled” among the various intermediate states. Indeed, there may be many distinct ways of cycling around a given mechanism or partial mechanism. Cycles associated with no net chemical change are called null cycles. All other cycles, by definition, involve net chemical change. For a null cycle, the product of all the equilibrium constants for the individual reaction steps in the cycle should equal unity. However, for any cycle associated with an overall reaction this product should equal the equilibrium constant for that reaction. This, the principle of detailed balance, follows from The Second Law of Thermodynamics and it provides an important constraint on the number of degrees of freedom permissible in assigning rate coefficients to the steps of a mechanism comprising one or more cycles.’ In this paper, we present a general method for explicit identification of the constraints imposed on the set of rate coefficients for a kinetic model by the requirement to satisfy detailed balance for all cycles in the model. The approach is exemplified by considering a hypothetical mechanism for a second order carriermediated transport process.

STRUCTURE-FUNCTION RELATIONS IN EF-HAND CA2+-BINDING PROTEINS: GENETIC ENGINEERING AND BIOPHYSICAL STUDIES OF BOVINE INTESTINAL CALCIUM PROTEIN (ICaBP)

Publisher Summary This chapter reviews the synthesis, expression and characterization by different biophysical techniques of four mutant bovine ICaBP:s and relate their properties to those of the wild type protein. To facilitate the construction of ICaBP:s with amino acid substitutions and deletions in the N-terminal half of the molecule containing the pseudo EF-hand Ca 2+ binding site a truncated version of the gene in which a 101 base pair EcoRI-PstI fragment corresponding to the first 33 amino acids of the protein was replaced by a polylinker made up from two oligonucleotides. Calcium binding constants were determined in Ca 2+ free 2 mM Tris–HCl buffer at pH 7.5 by three different methods to cover the large range of affinities displayed by the proteins. In the 1 H and 43 Ca NMR studies, the freeze dried protein was dissolved in D20 to a concentration of ca. 1 mM and pH adjusted to 8.0. The microcalorimetric measurements were made at 25°C using an LKB Batch Microcalorimeter equipped with an LKB titration assembly mounted on the outside of the calorimeter block. Although the 1 H NMR spectra of wild-type and mutant proteins by and large are very similar there are distinct differences among the spectra during the Ca 2+ titrations.