Significant improvement of signal-to-noise ratio in capillary electrophoresis through optimization of aperture width for UV absorption detection.

In capillary electrophoresis (CE), light flux passes through a capillary cell and is in most cases detected photometrically. Due to the thinness of the cell, a part of the light passes through the wall and misses hitting the sample. In most CE apparatuses, incident light is focused by converging lenses in order to condense light beams passing through the capillary. Considering the aberration of lenses and lens effects of capillary, we assumed that light beams inside were approximately parallel. Although the path lengths of light beams vary depending on their tracks, we could estimate the virtual light path length, L, by measuring absorbance when concentration and molar absorptivity of the sample solution were known. A light-restricting device consisting of narrow slits makes effectively L longer and signal intensity higher. On the other hand, noise increases as light width narrows. The signal-to-noise ratio showed a maximum at 68 microm of light width for a capillary with diameter of 75 microm. The optimized L was evaluated by the simulation. The experimental data verified it even in indirect UV detection. Our approach could help to design the optics of CE apparatuses.

Analysis of compounds containing carboxyl groups in biological fluids by capillary electrophoresis.

Capillary electrophoresis (CE) is one of the suitable separation techniques used to analyze drugs or metabolites in complicated sample matrices such as plasma, serum and urine. It sometimes requires only a simple process of sample pretreatment, deproteinization, dilution or extraction for biological fluids, otherwise no pretreatment is necessary. Various metabolic disorders concerning the compounds which possess carboxyl groups such as organic acids have been monitored by CE. Drug metabolism in the body can be monitored by the same technique. Recent publications suggest the feasibility of an automated system for diagnosis based on CE technique.

Method for detection of epsilon-secondary structure in the precore region of human hepatitis B virus DNA using a fluorescence-based polymerase chain reaction-single-strand-conformation polymorphism technique with capillary electrophoresis.

A portion of the precore region of the human hepatitis B virus (HBV) genome is the signal sequence with an epsilon secondary structure, which plays a role in the encapsidation of HBV pregenome RNA. To determine the genetic mutations which occur in the precore region of HBV, we have devised a typing method using a fluorescence-based polymerase-chain-reaction-single-strand conformation polymorphism technique with automated capillary electrophoresis (CE-FSSCP). Using the cloning sequencing method, we analyzed serum samples from 10 patients with hepatitis B, and detected three types of HBV-DNA including two mutants which are crucial to the function of the encapsidation sequence: position 1896 G (guanine) to A (adenine, stop codon), position 1899 G to A, and wild-type. We performed CE-FSSCP analysis of these three types of HBV-DNA and described conditions for determination of the mutations which play roles in the encapsidation of the HBV pregenome. The two types of epsilon mutants and wild-type DNA were identified as separate individual peaks respectively. The observed migration times of the three types of DNAs agreed fairly well with estimates obtained from total RNA secondary structure energy.

Fluorescence-based polymerase chain reaction-single-strand conformation polymorphism analysis of p53 gene by capillary electrophoresis.

Mutation of the p53 gene plays an important role in neoplastic progression in human tumorigenesis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) techniques are now available for the detection of point mutations. The original method using polyacrylamide gel electrophoresis is disadvantageous, particularly for clinical tests and for analysis of large numbers of samples. Therefore, using an automated capillary electrophoresis (CE) technique with a molecular-sieving polymer solution, we have devised a completely automatic fluorescence-based PCR-SSCP system (CE-FSSCP) for the differential detection of point mutations that dose not require SSCP with radioisotopes and polyacrylamide gels. The automatic CE-FSSCP system was developed for reproducible operations in the denaturation of double-stranded DNA and electrophoresis of single-stranded DNA. The detection system consists of a 100 W I2 lamp and photomultiplier. We performed CE-FSSCP with a 2% linear polyacrylamide polymer solution containing 5% glycerol. Four tissue specimens of lung tumors with mutations in exon 7 of the p53 gene were found to have mutant alleles; six-base-pair deletion at codons 247-248, a one-base-pair deletion at codon 260, a one-base-pair deletion at codon 244 and a GGC to CGC substitution at codon 244. We expect this technique to prove useful for the clinical DNA diagnosis of human cancers, determination of the therapeutic effect of anticancer agents and for the study of the molecular aspects of the mechanisms involved in the pathogenesis of human cancers.

Determination of trace amounts of albumin in human bronchoalveolar lavage fluids by fluorometry with chromazurol S.

Fluorometry using chromazurole S (CAS) was applied to determine trace amounts of albumin in human bronchoalveolar lavage fluids (BALF). The calibration curve was linear in the range of 5-60 micrograms/ml of albumin. The CAS method was proven to be much more selective for albumin than for IgG. Freezing of BALF samples did not affect albumin analysis by the CAS method after storage at -20 degrees C for 80 days. This finding suggests that albumin in the BALF samples is stable under these conditions. The correlation was highly linear (r = 0.966) between the albumin levels in concentrated BALF samples (n = 47) determined by the CAS method and by radial immunodiffusion. The CAS method is sensitive enough to determine albumin levels in unconcentrated BALF samples, whereas radial immunodiffusion often requires concentration. The former method is more suitable for measuring albumin in BALF samples than the latter, because concentration by ultrafiltration results in poor reproducibility. The concentration of albumin in BALF samples of healthy volunteers (n = 5) and patients with sarcoidosis (n = 32) was determined by the CAS method. There was a statistically significant difference (P < 0.01) in the albumin levels in BALF samples between healthy subjects and patients with sarcoidosis at a clinically active state (n = 15). This finding shows that the determination of albumin levels in BALF samples is useful for investigating lung diseases and that the CAS method is promising in the determination of trace albumin in BALF samples, because it is simple, sensitive and precise.

In vivo measurement of tumor blood oxygenation by near-infrared spectroscopy: immediate effects of pentobarbital overdose or carmustine treatment.

Near-infrared (NIR) spectroscopy was used to measure blood oxygen saturation (SO2) in vivo, in normal rat brain and in subcutaneously-implanted rat 9L gliosarcoma. Changes in cranial and tumor blood SO2 were measured during lethal pentobarbital overdose. After sacrifice, SO2 of cranial blood fell rapidly to a mean of 5.0% of the pre-sacrifice values, whereas SO2 of tumor blood stabilized at a mean of 72.4% of the pre-sacrifice values. This suggests that oxygen consumption by tumor is very low compared to brain. Cranial blood had a higher SO2 than tumor blood before sacrifice (p = 0.03), and a lower SO2 after sacrifice (p = 0.02). The magnitude of the change in SO2 after sacrifice was greater in normal brain than in tumor (p = 0.02), showing that brain tissue uses a greater proportion of the oxygen in ischemic blood than does tumor tissue. To determine the effect of carmustine (BCNU) treatment on tumor and cranial blood SO2, we compared BCNU-treated rats with sham-treated rats. Continuous NIR measurements before and immediately following treatment (ie. over 30-60 min) showed that tumor blood SO2 tended to increase after BCNU treatment, whereas SO2 tended to decrease following sham-treatment. The difference in SO2 between treated and control tumors was significant at 60 min (p = 0.02). Thus BCNU treatment can potentially result in immediate increases in tumor oxygenation. The increase in treated tumor blood SO2 occurred despite the fact that there was no change in cranial blood SO2 even at day 4 following treatment. Tumor blood SO2 was inversely correlated with tumor size (p = 0.001), confirming that blood is more poorly oxygenated in large tumors.

Cryoenzymic studies on actomyosin ATPase: kinetic evidence for communication between the actin and ATP sites on myosin.

The post-ATP binding steps of myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) ATPases were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The cleavage and release of Pi steps were studied by the rapid-flow quench method and the interaction of actin with S1 plus ATP by light scattering in a stopped-flow apparatus. At -15 degrees C, the interaction of actin with S1 remains tight, and the Km for the activation of S1 ATPase is very small (0.3 microM). The chemical data were interpreted by E + ATP----E*.ATP----E**.ADP.Pi----E*.ADP----products, where E is S1 or actoS1. In Pi burst experiments with S1, there was a large Pi burst of free Pi, but E**.ADP.Pi could not be detected. Here the predominant complex in the seconds time range is E*.ATP and in the steady-state E*.ADP. With actoS1, there was a small Pi burst of E**.ADP.Pi, evidence that the cleavage steps for S1 and actoS1 are different. From the stopped-flow experiments, the dissociation of actoS1 by ATP was complete, even at actin concentrations 60X its Km. Further, no interaction of actin with the key intermediate M*.ATP could be detected. Therefore, at -15 degrees C, actoS1 ATPase occurs by a dissociative pathway; in particular, the cleavage step appears to occur in the absence of actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient kinetics of oxygen dissociation from ferrous subunits of iron-cobalt hybrid hemoglobins. The principal reaction controlling the co-operativity.

The oxygen dissociation constants from Fe subunits in the half-ligated intermediate states of Fe-Co hybrid hemoglobins, alpha(Fe-O2)2 beta(Co)2 and alpha(Co)2 beta(Fe-O2)2, have been determined as functions of pH, temperature and inositol hexaphosphate. The oxygen dissociation rates from alpha(Fe-O2)2 beta(Co)2 are estimated to be more than 1300 s-1 for the deoxy quaternary state (T-state) and less than 3 s-1 for the oxy quaternary state (R-state) at 15 degrees C in 50 mM-Tris or Bis-Tris buffer containing 0.1 M-Cl-, while those of alpha(Co)2 beta(Fe-O2)2 are more than 180 s-1 and less than 5 s-1 for the T and R-states, respectively. The pH dependence of the oxygen dissociation rate from Fe subunits is large enough to be accounted for by the R-T transition, and implies that those half-ligated intermediate hybrids mainly exist in the R-state at pH 8.8, and in the T-state at pH 6.6, while other studies indicated that the half-ligated hybrids are essentially in the R-state at pH 7. Large activation energies of the oxygen dissociation process of 19 to 31 kcal/mol determined from the temperature dependence suggest that the process is entropy-driven.

Electron paramagnetic resonance study on crosslinked asymmetric Fe(II)-Co(II) hybrids of hemoglobin.

Asymmetric Fe-Co hybrid hemoglobins (Hbs) containing one, two, and three Co(II) protoporphyrins IX per tetramer have been synthesized. These asymmetric Fe-Co hybrids have been prepared by appropriate combinations of subunits of HbA, HbS, Co(II)HbA, Co(II)HbS, and Fe-Co hybrid Hbs and crosslinking with bis(3,5-dibromosalicyl)fumarate, followed by purification with molecular sieve and ion-exchange chromatography. Electron paramagnetic resonance (EPR) spectra have been measured for the crosslinked asymmetric Fe-Co hybrids to investigate the intermediate ligation states of Hb. The electronic state of the Co(II) ion provides a useful probe to estimate how ligation to iron-containing subunits changes the structure of the cobalt-substituted heme vicinity as described previously (T. Inubushi and T. Yonetani (1983) Biochemistry 22, 1894-1900). The crosslink does not affect the EPR spectra of Fe-Co hybrids. EPR spectra of the alpha (Co) subunits in crosslinked hybrids in the fully deoxy state exhibit a mixture of a broad EPR component and a narrow EPR component which correspond to distorted and undistorted Co(II) coordinate states, respectively, and the ratio of the components varies with pH. EPR spectra of the di-liganded asymmetric hybrid, [alpha (Co) beta (Co)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, and tri-liganded hybrid, [alpha (Co) beta (Fe-CO)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, show that the coordination state of the alpha (Co) ion in these hybrids is changed to the completely undistorted state in the pH range between 6.5 and 8.8 upon ligation of carbon monoxide (CO). Mono-liganded hybrids, [alpha (Co) beta (Fe-CO)]A-[alpha (Co) beta (Co)]sXL and [alpha (Fe-CO) beta (Co)]A-[alpha (Co) beta (Co)]sXL show intermediate EPR spectra at pH 6.5-7.6, which represent a mixture of the broad and narrow EPR components. The broad component is converted into the narrow one more extensively upon the ligation to the beta (Fe) subunit than to the alpha (Fe) subunit. However, the coordination state of an alpha (Co) subunit in mono-liganded hybrids corresponds to a completely undistorted coordination structure at pH 8.2-8.8. The EPR signal of the alpha (Co) ion is sensitive to the release of alkaline Bohr proton(s) and consequently to the allosteric quaternary structural change of Hb.

Kinetic study on the interaction of Rhizopus chinensis aspartic protease with Streptomyces pepsin inhibitor (acetylpepstatin).

The fluorescence of tryptophan residues of Rhizopus chinensis aspartic protease was quenched about 25% upon binding with an inhibitor, Streptomyces pepsin inhibitor (acetylpepstatin). The kinetics of binding between the enzyme and the inhibitor was studied by the fluorescence stopped-flow method. The concentration dependence of apparent rate constants was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow unimolecular process. The unimolecular process was interpreted to be a conversion from a transient intermediate to the final complex in which the inhibitor is tightly bound to the active site of the enzyme. Fluorescence quenching occurred essentially in the unimolecular process, which suggests microenvironmental transition around at least one tryptophan residue in the enzyme-substrate complex.

The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.

Kinetic studies on the binding of gostatin, a suicide substrate for aspartate aminotransferase, with the isoenzymes from porcine heart mitochondria and cytosol.

The reaction of pig heart mitochondrial and cytosolic aspartate aminotransferases (abbreviated to mAspAT and cAspAT, respectively) with an enzyme-suicide substrate (mechanism-based inhibitor), gostatin (5-amino-2-carboxyl-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid) was studied kinetically, by following the spectral change with a micro-stopped-flow apparatus, as well as the inactivation of the enzyme activity. No significant difference in kinetic behavior was observed between mAspAT and cAspAT. From the analysis of time-dependent spectral change, no positive evidence for the existence of spectrophotometrically distinguishable intermediates was obtained. Both the spectral change and the inactivation followed, at least in appearance, simple bimolecular association kinetics, under the conditions studied. However, the second-order rate constant of the spectral change was found to be 1.5 to 2 times as large as that of the inactivation. The effects of pH and temperature on k(on) (the second-order rate constant of the spectral change) were also studied.

Binding between thermolysin and its specific inhibitor, N-phosphoryl-L-leucyl-L-tryptophan (PLT).

The interaction between thermolysin and its specific inhibitor, PLT (N-phosphoryl-L-leucyl-L-tryptophan), has been investigated by steady-state inhibitory kinetics analysis, fluorometric titration, and the stopped-flow method. The inhibitor constant of PLT, Ki, and the dissociation constant of thermolysin(E)-PLT(I) complex, Kd, are found to be smaller by a factor of 4 to 300, depending on pH, resulting in stronger binding, than those of talopeptin and phosphoramidon, but all of them show similar pH dependence. The dependence of the apparent first-order rate constant, Kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular step followed by a slow unimolecular step, (Formula: see text). The values of K-1 (the dissociation constant of the intermediate EItr) and K-2 (the backward rate constant in the unimolecular step) are not so significantly different between PLT and talopeptin, while the K+2 (forward rate constant in the unimolecular step) value for PLT is about 14 times larger than that of talopeptin (pH 5.5). These facts suggest that the forward rate of the isomerization step, EItr----EI, is much larger in the absence of the sugar moiety of talopeptin, and hence it induces the stronger binding of PLT to thermolysin than that of talopeptin.

Binding between thermolysin and its specific inhibitor, phosphoramidon.

Equilibrium and kinetic studies on the interaction between thermolysin (E) and its specific inhibitor (I), phosphoramidon (N-(alpha-L-rhamnopyranosyloxyphospho)-L-leucyl-L-tryptophan), have been made by steady-state inhibitory kinetics analysis, fluorometric titration and the stopped-flow method. The inhibitor constant, K1, the dissociation constant of the El complex, Kd, directly obtained by fluorometric titration, and the apparent second-order association constant, kon, obtained with the stopped-flow method are very similar to those for talopeptin (Kitagishi, K., et al. (1983) J. Biochem. 93, 47-53 and 55-59), whose molecular structure differs from that of phosphoramidon only in the configuration of the OH group at the C-4 atom of the sugar moiety. The result suggested that the OH group is not essential for the binding to thermolysin.

Studies on the chemical modification of tryptophan residues in thermolysin and in talopeptin (MKI) with N-bromosuccinimide.

Tryptophan residues in thermolysin (3 Trp/molecule) and in its specific inhibitor, talopeptin (1 Trp/molecule), were modified with N-bromosuccinimide (NBS). The decrease in the absorption at 280 nm and the fluorescence intensity above 310 nm (excited at 280 nm) accompanying the modification were followed by the stopped-flow method as a function of time. When the sole tryptophan residue of talopeptin was modified with NBS, its inhibitory activity against thermolysin was almost completely destroyed. For thermolysin, the decrease in molar absorptivity corresponds to the modification of one of its three tryptophan residues, and the enzyme activity does not decrease significantly with the modification (remaining activity was 96% at [NBS]/[E] = 6). The results obtained for the modification of EI complex suggested that the formation of EI complex remarkably reduces the rate constant for the modification of the tryptophan residue in talopeptin, but does not affect that of the tryptophan residue(s) in thermolysin.

Binding between thermolysin and talopeptin (MKI) in which the tryptophan residue was converted into kynurenine.

The tryptophan residue of talopeptin, which is a specific inhibitor for thermolysin, was converted into kynurenine by ozonization followed by acid-catalyzed hydrolysis, and (Trp leads to Kyn) talopeptin (Kyn-talopeptin) thus obtained was purified with gel-chromatography. The inhibitor constant of Kyn-talopeptin, K1, and the dissociation constant of thermolysin-Kyn-talopeptin complex, Kd, directly obtained by fluorometric titration were in good agreement with each other. These values were found to be about 10 times larger than those of intact talopeptin, but both inhibitors showed a similar pH dependence. Upon the binding of Kyn-talopeptin with thermolysin, the protein fluorescence of thermolysin decreases by about 60%, while the kynurenine fluorescence (measured at 450 nm when excited at 360 nm) of the inhibitor increases about 14 times. The measurements of the excitation and fluorescence spectra of EI complex strongly indicated the energy transfer from tryptophan residue(s) (the donor) of the enzyme to kynurenine residue (the acceptor) of the inhibitor. The distance between the donor and the acceptor was roughly estimated to be 18 A. This value is in good agreement with the one expected from the crystallography of phosphoramidon-thermolysin complex. The binding process was studied kinetically with the stopped-flow method over the pH range between 4.5 and 8.5, by monitoring the decrease in the fluorescence intensity of the enzyme tryptophan caused by the complex formation. Comparison of the data with those previously obtained for talopeptin-thermolysin system revealed that the replacement of the tryptophan residue by kynurenine of the inhibitor does not affect the apparent second-order association rate constant, kon, seriously.