No abstract

Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.

The hydrodynamic properties of purified D-ßhydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme isolated from bovine heart mitochondria, have been studied using the active-enzyme sedimentation technique. An active complex of BDH with dioctanoyl-L-a-phosphatidylcholine, PC(8:0), was sedimented under assay conditions, and the sedimenting boundary was followed by observing the reduction of NAD+. Between 2.4 and 30 µg of BDH/mL, the ¿ 2o,w increased and was inversely related to the enzymatic activity. The most active species has the smallest apparent s2o,w (3.3 ± 0.2 S). The diffusion coefficient of the BDH-PC(8:0) complex at 2.4 jug/mL, measured by the free diffusion method, was found to be 4.9 ± 0.5 X 10-7 cm2/s; at higher protein concentrations, Z)2o,w decreased. The hydrodynamic studies both of the active BDH-PC(8:0) complex and of the apoen-^Biological membranes are macromolecular complexes consisting of proteins and phospholipids. D-/3-Hydroxybutyrate dehydrogenase, normally bound to the mitochondrial inner membrane, has been purified from bovine heart mitochondria to homogeneity as a soluble, phospholipid-free protein, apo-