Masaru Sogami

Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy

Conformational changes in cystine disulfide bridges of bovine serum albumin during acid‐induced isomerization (N→F and F→E transitions) have been studied with Raman spectroscopy. In an X‐ray crystallographic study of human serum albumin, Carter and Ho reported that all disulfide bridges of the albumin molecule are in the gauche‐gauche‐gauche conformation [1] . On the other hand, the solution structure of bovine serum albumin examined by Raman spectroscopy differs from its crystal structure in the conformation of some of the disulfide bridges. Two Raman bands were detected at 520 and 505 cm−1 in the disulfide stretching mode region, suggesting that the 17 disulfide bridges in the N‐form of bovine serum albumin solution take both the gauche‐gauche‐gauche and gauche‐gauche‐trans conformations. The ratio of the peak intensities at 520 and 505 cm−1 (I505/I520) is increased from 1.6 to 2.1 and from 2.1 to 6.3 on going from the N‐ to the F‐form and from the F‐ to the E‐form, respectively, indicating that the gauche‐gauche‐trans conformation of the disulfide bridges is converted to a gauche‐gauche‐gauche one which is the most energetically stable form during the acid‐induced isomerization. However, small amounts of gauche‐gauche‐trans conformation still remain even in the E‐form.

Increased oxidized form of human serum albumin in patients with diabetes mellitus

High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on Asahipak GS-520H columns at neutral pH (6.87) showed a clear resolution of human mercaptalbumin (HMA) and nonmercaptalbumin (HNA), which are reduced and oxidized form of HSA, respectively. We studied the conversion of HMA to HNA (mercapt-nonmercapt conversion) as an index of oxidative change of the tissues and organs in 28 normal subjects and in a total of 47 patients with non-insulin dependent diabetes mellitus (NIDDM). Mean (+/- SD) values of the HMA fraction of HSA, f(HMA), [HMA/(HMA + HNA)], was significantly lower in NIDDM patients than in normal subjects (0.63 +/- 0.067 vs 0.75 +/- 0.028, P < 0.001). It was lower in poorly controlled NIDDM patients (0.63 +/- 0.058, n = 20) than in well controlled NIDDM patients (0.67 +/- 0.032, n = 9) (P < 0.05). Plasma glucose values sampled on occasions including overnight fasting and postprandial ones (r = -0.441, n = 47, P < 0.01), but not plasma glucose values sampled on overnight fasting (r = -0.345, n = 29) or postprandial (r = -0.467, n = 18) conditions and HbA1c (r = -0.211, n = 34), negatively correlated with the f(HMA) values, indicating that mercapt-nonmercapt conversion may not be due to cumulative hyperglycemia over a month, but due to short-term alteration in blood glucose level. The presence or absence of diabetic complications including nephropathy, retinopathy and neuropathy did not affect the f(HMA) values. In conclusion, decreased f(HMA) values in the diabetic patients suggested the presence of a rapidly altered oxidative change of albumin due to hyperglycemia.

High-performance liquid chromatographic studies on non-mercapt α mercapt conversion of human serum albumin. II

Abstract High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on a GS-520 column with 0.03 M sodium phosphate buffer—0.15 M sodium sulphate (pH 6.87) showed three peaks, the principal component corresponding to human mercaptalbumin (HMA) and the secondary and tertiary components to nonmercaptalbumin (HNA). Using HPLC analysis, the nonmercapt → mercapt conversion of HSA during haemodialysis and the mercapt → nonmercapt conversion after haemodialysis in chronic renal failure were re-confirmed, indicating that HMA is a covalent carrier protein for sulphur-containing amino acids. Fractions of HMA in various liver diseases were significantly lower than those of healthy male adults.

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt<-->nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical_ patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA+HNA)]+/-standard deviation, fHMA+/-sigma], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were 0.636+/-0.050 (n = 83) and 0.615+/-0.062 (n = 14), respectively. fHMA values were markedly lower than those for healthy male adults of 0.750+/-0.028 (n = 28). fHMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in cardiac surgical patients. Although fHMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients never recovered even on the 21st postoperative day.

Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions.

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.

Magnetic circular dichroism on oxygen complexes of hemoproteins Correlation between magnetic circular dichroism magnitude and electronic structures of oxygen complexes

Magnetic circular dichroism (MCD) and natural circular dichroism (CD) spectra are reported for horseradish peroxidase Compounds II and III, and kangaroo myoglobin Compound II at pH values of 8.5 and 4.9. These compounds exhibited MCD spectra of apparent Faraday A term both in the Soret and Q regions, except for myoglobin compounds in the Soret region where intrinsic temperature dependence showed large contribution from Faraday C terms. Comparison of these data with the MCD spectra of the dioxygen complexes of hemoglobin (myoglobin) and cytochrome P-450 revealed that the magnitude of the apparent Faraday A term trough at the Q0-0 bands decreased in the order of O2 complexes of hemoglobin (myoglobin) ([theta]M not equal to 16) greater than horseradish peroxidase Compound III ([theta]M not equal to 8) greater than O2 complex of cytochrome P-450 ([theta]M not equal to 4). The [theta]M values of the oxygen complex of cytochrome P-450 is similar to those observed for the compounds II of horseradish peroxidase and kangaroo myoglobin. From these observations it was concluded that the magnitude of MCD, especially the trough depth of the Q0-0 band, has direct correlation to the electronic states of the oxygen complexes of the hemoproteins. The implication of the findings was discussed in terms of the iron electronic structures perturbed by the axial ligation.

Fluorimetric studies on the structural transition of bovine plasma albumin in acidic solutions

The acid-induced isomerization (the NF transition) and expansion of bovine plasma albumin were studied by measuring [α]233 and tryptophyl fluorescence. Most of the changes in tryptophyl fluorescence intensity at 350 nm correlated exactly with the NF1 transition and/or the initial part of the NF transition.

The kinetic studies on the intramolecular SH, S-S exchange reaction of bovine mercaptalbumin

Bovine mercaptalbumin (BMA) has 17 disulfide bonds and one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction (N-A isomerization, molecular aging) in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). The aging reaction was completely reversible and strongly affected by environmental factors, such as pH, temperature, ionic strength, Ca2+, nonbranched short-chain fatty acids, etc. Disulfide configuration (or pairing of disulfide bonds) was affected by the environmental factors. Obtained results might support the concept of Klotz (1966) that protein conformation (or three-dimensional structure) is dependent upon (i) the primary structure and (ii) constituents of the solvent.

Equivalent cross-relaxation rate imaging in the synthetic copolymer gels and invasive ductal carcinomas of the breast

The values of equivalent cross-relaxation rate (ECR) correlated well with [i] water conditions in various copolymer gels and [ii] nature of malignant cells with regard to nuclear dysplasia and mitotic potential in breast carcinomas. The synthetic copolymer gels composed of any two or three monomers among 2-hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA), N-vinyl-2-pyrrolidinone (N-VP), methyl methacrylate (MMA) and benzyl methacrylate (BMA). The ECR measurement was performed by using an off-resonance saturation pulse under conventional field-echo imaging at frequency within +/- 75 ppm apart from the water resonance frequency. The ECR values were readily to determine and non-time consuming parameter for cross relaxation rate. The ECR values at the frequency offset by 7-ppm (ECR-7) were divided the sample gels two classes, which must correspond to hydrophilic or hydrophobic ones. The sensitivity in the gels was nearly equivalent to the cross-relaxation rate itself. In the breast carcinomas, the ECR-7 correlates with the nature of malignant cells with regard to nuclear dysplasia and mitotic potential. The ECR-7 is better or more accurate than the STR-7 because the SDNRs between carcinoma and glandular tissue increased by approximately 50% on the ECR-7 compared with the STR-7. Thus the ECR values could be a new parameter for malignancy and cell proliferative activity of the breast carcinomas with non-invasive modalities by magnetic resonance imaging.

Saturation transfer ratio imaging in invasive ductal carcinomas of the breast

A prospective study was performed to investigate the correlations between saturation transfer ratio (STR) and histologic parameters of invasive ductal carcinomas in human breast. The histologic parameters investigated were the extent of fibrosis in the intercellular matrix, dysplastic changes of nuclei, and mitotic index. Twenty-seven patients with breast carcinoma were examined using an off-resonance saturation pulse in conjunction with conventional field-echo T(1)-weighted imaging at frequency offsets of 448 Hz and 1200 Hz from water resonance. The values of STR at frequency offset of 1200 Hz (STR(1200)) increased from non-scirrhous carcinoma to scirrhous carcinoma. Although STR(1200) showed correlation with the extent of fibrosis in the intercellular matrix (p<0.01, n = 27), they did not correlate with the dysplastic changes of nuclei or mitotic index. On the other hand, the values of STR at frequency offset of 448 Hz (STR(448)) demonstrated close correlation to dysplastic changes of nuclei and mitotic index (p<0.01, n = 27). STR(1200) correlates with the structural characteristics and STR(448) correlates with the nature of malignant cells with regard to nuclear dysplasia and mitotic potential.