Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.

Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein.

Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase [EC 2.5.1.1, EC 2.5.1.10]. The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants. A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences. A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human. The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities. Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts.

The effect of label affinity on the sensitivity and specificity of a hapten radioimmunoassay: a comparison of three [125I]diphenylhydantoin radioligands with the 14C-labelled drug.

The effect on the sensitivity and specificity of a radioimmunoassay for diphenylhydantoin (DPH)has been investigated using three 125I-labelled tyrosine ester derivatives of DPH having different bridge lengths between the tyrosine moiety and the DPH moiety and 14C-labelled DPH. The results demonstrate that for a hapten which does not completely fill the antibody-binding sites, greatest sensitivity is achieved when the bridge of the iodine label is most dissimilar to that present in the original immunogen, when the hapten and label affinities are nearly equivalent. Greatest specificity is achieved with the label which most resembles the original immunogen. These results illustrate the difficulty of designing satisfactory labels for assays of both high specificity and sensitivity since minimal changes in label structure may produce greatly amplified changes in the subsequent affinity of the label for the antiserum.