Kazuo Satake

Copper complexes at N- and C-site of ovotransferrin: Quantitative determination and visible absorption spectrum of each complex

Copper complexes at the two sites of ovotransferrin (TF) differed markedly in the rate of Cu release by EDTA. During the reaction, lambda max of the remaining Cu-Tf complex shifted to red side, while the difference spectrum of FenCu2-nTf vs. FenTf in which the N-site had been preferentially occupied with Fe had lambda max at blue side from that of Cu2Tf, 440 nm. From these results, the intrinsic spectrum for Cu-complex at each site was assigned: lambda max 450 nm for N- and 430 nm for C-site. The differences in the release rate and the spectrum can be used for the identification of the two domains of Tf and for the analysis of metal-binding behavior of each site.

Preparation and characterization of trypsin‐nicked ovotransferrin

The N‐ and C‐domains isolated from ovotransferrin (Tf) with trypsin could be separated from each other and from intact Tf by HPLC with a TSK‐GEL G‐3000SWG‐0.1% SDS system. The analytical method revealed that Fe (III)‐saturated Tf (Fe2Tf) of 77 kDa was hydrolyzed by trypsin preferentially at the portion connecting both domains. The main product was a nicked Fe2Tf, in which the two fragmented domains of 35 kDa each were still bound together non‐covalently and showed a notable cooperativity on their denaturation.

Binding of Cu (II), Tb (III) and Fe (III) to chicken ovotransferrin

The kinetics of binding of Cu (II), Tb (III) and Fe(III) to ovotransferrin have been investigated using the stopped-flow technique. Rate constants for the second-order reaction, k+, were determined by monitoring the absorbance change upon formation of the metal-transferrin complex in time range of milliseconds to seconds. The N and C sites appeared to bind a particular metal ion with the same rate; thus, average formation rate constants k+ (average) were 2.4 × 104 M−1 s−1 and 8.3 × 104 M−1 S −1 for Cu (II) and Tb (III) respectively. Site preference (N site for Cu (II) and C site for Tb (III)) is then mainly due to the difference in dissociation rate constant for the metals. Fe (III) binding from Fe-nitrilotriacetate complex to apo-ovotransferrin was found to be more rapid, giving an average formation rate constant k+ (average) of 5 × 105 M−1 s−1, which was followed by a slow increase in absorbance at 465 nm. This slow process has an apparent rate constant in the range 3 s−1 to 0.5 s−1, depending upon the degree of Fe (III) saturation. The variation in the rate of the second phase is thought to reflect the difference in the rate of a conformational change for monoferric and diferric ovotransferrins. Monoferric ovotransferrin changes its conformation more rapidly (3.4s−1) than diferric ovotransferrin (0.52 s−1). A further absorbance decrease was observed over a period of several minutes; this could be assigned to release of NTA from the complex, as suggested by Honda et al. (1980).

Development of Novel C-Terminal Sequencing Methods

Development of a reliable C-terminal sequencing method has been expected from various aspects including sequencing protein, analyzing posttranslational process, confirming recombinant proteins and cloning. Carboxypeptidase digestion has been commonly used with limitations. Since a classical isothiocyanate degradation was proposed, several modifications were reported in the past two conferences (Hawkes and Boyd, 1991; Inglis et al., 1991; Miller and Shively, 1989). The present paper summarizes two related novel carboxy-terminal sequencing methods using perfluorinated carboxylic acids in monohydrate form and carboxylic acid anhydrides. These reagents produce the mixtures of C-terminal successive degraded molecules which are able to be analyzed by fast-atom-bombardment (FAB)- or electrospray ionization (ESI)-mass spectrometry (MS).

Kinetic study on oxygenation of Lingula unguis hemerythrin using the stopped-flow 02-jump method

AbstractO2-jump experiments with an improved stopped-flow apparatus have been used to study oxygenation and deoxygenation processes in Lingula unguis hemerythrin. With an O2 electrode set in the observation cell, O2 concentration conld be obtained directly. The reliability of this method has been compared with other conventional methods.O2-jump (up and down) experiments were carried out with L. unguis hemerythrin at pH 6.8 (non-cooperative pH) and at pH 7.6 (cooperative pH). At pH 6.8, both O2-jump (up) and O2-jump (down) experiments showed single exponential processes which were consistent with the following scheme:

Removal of N‐terminal formyl groups and deblocking of pyrrolidone carboxylic acid of proteins with anhydrous hydrazine vapor

{\text{Hr + O}}_{\text{2}} \mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{off}}} }^{{\text{k}}_{{\text{on}}} } {\text{HrO}}_{\text{2}}

Reaction of Pentafluoropropionic Anhydride Vapor on Polypeptide as Revealed by Mass Spectrometry. A Carboxypeptidase Mimetic Degradation

. The value of kon was estimated to be (4.4 ± 0.5) × 105 M−1 s −1, and koff was (15 ± 5) s−1. These values are consistent with those obtained by the temperature-jump method (Zimmer et al. 1986). At pH 7.6, O2-jump (up) experiments showed two relaxation processes, whereas O2-jump (down) experiments showed a single exponential process. The faster process in the O2-jump (up) experiments could be attributed to the same process as that seen in the temperature-jump experiments (Zimmer et al. 1986). The slower process in the O2-jump (up) experiments corresponds to the process obtained in the O2-jump (down) experiments. The results are discussed in terms of a state with intermediate affinity in O2-binding and with the possible existence of a slow step in O2-binding.

BACTERIAL OXIDATION OF SOME PRIMARY AMINES

The amino acid sequence of a protein of interest is usually one of the first pieces required in today’s molecular biology, be it for gene cloning or synthesis of immunoreactive peptides. To date, amino(N)-terminal sequencing using the Edman degradation procedure has almost exclusively provided such data. Methodologies for sequencing proteins from their carboxy(C)-termini have remained relatively primitive requiring much protein in return for little sequence information. Carboxypeptidase digestion is still the most widely used method despite its intrinsic limitations of substrate specificity and endoprotease contamination. Several chemical degradation methods have been reported (Stark, 1968; Yamashita,1971; Bailey et al., 1994), and a few automated C-terminal sequencers are almost available to the public.