David A. Deranleau

Wavelength dependence of the zero-field splittings in the triplet state of tryptophan☆

Abstract It is shown that abrupt discontinuities occur in the zero-field splitting parameters of the isolated amino acid, tryptophan, and its derivatives as a function of the wavelength of the optically detected emission from the excited triplet state. These discontinuities are attributed to the convolution of the magnetic transitions associated with each of the elementary vibronic components assuming that the frequency of the magnetic transition is a linear function of the wavelength for each elementary vibronic component.

A recording fluorescence polarization photometer

Abstract An instrument is described for the automatic recording of the polarization of fluorescence of solutions as a function of wavelength or time. A splitbeam optical system is employed to measure the intensity of the components of the polarized emission vibrating parallel and perpendicular to the direction of propagation of the exciting light. A small, highly accurate analog computer is used to generate a variety of functions from the two signals, and the results are presented on an XY plotter. The accuracy of the polarization measurements is believed to be of the order of 1% on the basis of comparison with an independent method.

[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.

Effects of pH on the exposure of aromatic donor residues in solution topography studies of bovine trypsin by charge transfer

Abstract Fully exposed aromatic amino acid side chains on the surface of proteins can act as donors in the formation of π D −π A ∗ charge transfer complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride (1-methylnicotinamide chloride) (Hinman et al ., 1974). A study of the surface aromatic donor residues of native trypsin in the pH range 3 to 9 indicates that two of the four tryptophan and about five of the ten tyrosine residues are sufficiently exposed to bind MNCl † and give rise to characteristic charge transfer absorption spectra. Between about pH 4 and pH 8, there is an increase in the overall charge transfer absorbance of the system that parallels the pH activity profile of the enzyme. An analysis of the charge transfer properties of the complex at both ends of the pH curve indicates that the observed change is due to an increase in the Trp-MNCl association constant of one of the two exposed tryptophan residues. A similar investigation of the pancreatic trypsin inhibitor-trypsin complex revealed a single available tryptophan residue whose association constant did not change with pH. Other chemical and physical evidence strongly suggests that access to the indole ring of Trp215 is blocked by the bound inhibitor molecule in the inhibitor-trypsin complex, thus implicating Trp215 as the residue with the pH-dependent association constant. If this interpretation is correct, the observed changes in the charge transfer properties of Trp215, whose peptide bond forms a part of the specificity pocket of the enzyme (Krieger et al. , 1974), could serve to monitor conformational rearrangements or flexibility in the region of the enzyme that is directly concerned with the binding of substrates. The implications of the charge transfer study are discussed in terms of the crystal structure models for diisopropylphosphorofluoridate and benzamidine-inhibited trypsins, and are compared with results obtained by other solution techniques used for probing tertiary structure.

The solution conformation of bovine carboxypeptidase a: Reaction with 2-hydroxy-5-nitrobenzyl bromide and N-methylnicotinamide chloride.

The recent completion of the amino acid sequence [l] and of the high resolution electron density map [2] of crystalline bovine carboxypeptidase A lead to a study of the position of various amino acid side chains of the enzyme in an aqueous environment. For this reason, the availability of the tryptophanyl residues of this enzyme have been examined by two methods: chemical modification by 2-hydroxy-%nitrobenzyl bromide (I-INBB) [3,4] and complex formation with N-methylnicotinamide chloride [ 151. These experiments revealed that the seven tryptophanyl residues present in the enzyme [ 1 ] are unavailable. However, in contrast to earlier reports, [3,4] modification of the protein by 2-hydroxy-S-nitrobenzyl bromide at pH 7.0 occurs rapidly and apparently specifically at the o-amino group, suggesting that under certain conditions, the specificity of 2-hydroxy-5nitrobenzyl bromide is wider than previously thought.

A stopped-flow laser turbidimeter for studying changes in the shape of cells stimulated by external agents.

Abstract A laser turbidimeter suitable for following rapid morphological changes which may accompany the stimulation of cells suspended in an adequate medium is described. Stopped-flow mixing is used to initiate the reaction and the changes in light transmission are followed with a fast recording technique. The apparatus has been used to examine the ADP-induced shape change reaction of human blood platelets.

Multiple Equilibria in Macromolecular Systems

The theory of multiple equilibria for binding of adsorbable molecules to macromolecules is extended to the case of polymerizing systems. The general result reduces to a simple form if no binding sites are excluded on polymerization. It is shown that nonlinear results may be obtained in a binding experiment, depending on the choice of variables, and a method of measuring macromolecular polymerization from a binding experiment is suggested.

A simplified method for the calculation of the Z-average molecular weight from ultracentrifuge data.

Abstract A modification of the method of Lansing and Kraemer for obtaining Z-average molecular weights from ultracentrifuge measurements under equilibrium conditions is described. By employing the differrential form of the equation normally used for homogeneous materials, in conjunction with numerical differentiation, the amount of computation involved in obtaiining a Z-average molecular weight is greatly reduced. The modified treatment yields essentially the same values of the Z-average as does the original treatment, and the computation error is slightly less in the modified method than in the original. It is also shown that the Z-average molecular weight at each point in the cell can be derived directly from the Archibald equation relating concentration to distance in the cell. Tentative molecular weight averages of 80 000 (Z-average) and 76 000 (weight-average) are assigned to a sample of crystalline yeast aldolase.

Time-dependent total extinction analysis for simple reactions involving intermediates

Abstract When the individual species in a simple series reaction A → B → C have different light absorption or scattering extinction coefficients, the progress curves obtained from extinction measurements are generally biphasic. If the rate constants are not too dissimilar, the curves can exhibit maxima or minima, or be s-shaped or monotonic. The shape of the curves depends mainly on the relative values of the extinction coefficients. A few simple rules are developed, which usually suffice to give important qualitative information concerning the nature of the intermediate.

Direct determination of the stoichiometry of charge transfer complexes

The stoichiometry of the charge transfer complex between N-acetyl-L-tryptophan and 1-methylnicotinamide chloride has been determined to be precisely 1:1 by direct measurement of the molecular weight of the complex. The result is of interest both in terms of a general method for determining the stoichiometry of charge transfer complexes, and in terms of the probable stoichiometry of specific charge transfer complexes between 1-methylnicotinamide chloride and the exposed tryptophyl side chains of certain proteins. In the latter case, the result provides experimental proof for the assignment of extinction coefficients of specified magnitudes to the homomorphic model complexes which serve as the basis for the interpretation of results with proteins.