Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

Synthetic formyl tripeptide chemoattractants: a C(alpha,alpha)-dialkylated, amphiphilic glycyl residue at position 1.

The two diastereomeric tripeptides f-(S)-HmMet-Leu-Phe-OMe and f-(R)-HmMet-Leu-Phe-OMe, analogues of the prototypical chemoattractant f-Met-Leu-Phe-OH, were synthesized in solution by classical methods and fully characterized. A conformational study was performed in solution by 1H-NMR. Concomitantly, the two peptides were tested for their ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The conformational and biological data are discussed with regard to the proposed model of the chemotactic receptor on neutrophils.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

One step immobilization of peptides and proteins by using modified parylene with formyl groups

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.     

The isokinetic relationship. VII. Statistical analyses and examples for unimolecular reaction systems

The existence of the isokinctic relationship (IKR), also called the ‘compensation effect’, is examined for unimolecular reaction series. A statistical analysis on the bases of a common point of intersection of the Arrhenius plots is given. This method is performed using three experimcntal examples. They exhibit highly precise IKRs, showing that this relationship is not necessarily an artefact based on measurement errors. The isokinetic tempcrature, T_iso, of a reaction series is significantly higher in an excess of a mono- atomic inert gas than in gas phase reactions without the inert gas or for reactions in condensed phase. The isokinetic temperature seems to correspond to energy quanta available in the system which can be found in the vibrational spectra of the system.     

Spectrophotometric resolution of ternary mixtures of tryptophan, tyrosine, and histidine with the aid of principal component-artificial neural network models

A simple and sensitive spectrophotometric method to resolve ternary mixtures of tryptophan (Trp), tyrosine (Tyr), and histidine (His) in synthetic and water samples is described. It relies on the different kinetic rates of the analytes in their oxidative reaction with potassium ferricyanide (K(3)Fe(CN)(6)) in alkaline medium. The absorbance data were monitored on the analytical wavelength (420 nm) of K(3)Fe(CN)(6) spectrum. Synthetic mixtures of the three amino acids were analyzed, and the data obtained were processed by principal component-artificial neural network (PC-ANN) models. After reducing the number of spectral data using principal component analysis, an artificial neural network consisting of three layers of nodes was trained by applying a back-propagation learning rule. Tangent and sigmoidal transfer function were used in the hidden and output layers, respectively. The analytical performance of this method was characterized by relative standard error. The method allowed the determination of Trp, Tyr, and His at concentrations between 10 and 55, 10 and 60, and 10 and 40 microg ml(-1), respectively. The results show that the PC-ANN is an efficient method for prediction of the three analytes.     

Association of dominant loci for resistance to Pseudomonas syringae pv. pisi with linkage groups II, VI and VII of Pisum sativum

Morphological characters, isoenzymes and recombinant inbred lines were employed to assign four loci for resistance to Pseudomonas syringae pv pisi to genetic linkage groups in Pisum sativum. A total of five morphological markers and 11 isoenzyme loci were screened in two independent F₂ P. sativum populations: Vinco × Hurst’s Greenshaft (V×HGS) and Partridge × Early Onward (P×EO). Mapping was also carried out in two recombinant inbred populations, unrelated to the F₂ populations. Previously reported linkage between resistance genes Ppi3 and Ppi4 was confirmed. Linkage was also detected between resistance gene Ppi2 and the isoenzyme locus Aldo (linkage group VII). The linked loci Ppi3 and Ppi4 were associated with a (linkage group II). A further resistance gene Ppi1 was associated with linkage group VI close to the hilum colour gene P1. RAPD markers tested in the cross P×EO were not well targeted; however, one marker, OPA-20_0.71, showed linkage to Ppi3. Received: 3 July 2000 / Accepted: 27 October 2000