¹H‐NMR and CD studies on the structural transition of serum albumin in the acidic region—The N→F transition

Abstract

Helical content (fα) of bovine mercaptalbumin (BMA) showed the characteristic two‐step decrease in the acidic region, one corresponding to the N→F transition (pH 4.40→3.75; fα, 0.68→0.58) and the other to the F→E transition (the acid‐expansion) (pH 3.60→2.90; fα, 0.58→0.48). However, fα of human serum albumin (HSA) mainly decreased in the N→F transition (N→F, pH 4.6→3.4; fα, 0.70→0.55 and F→E, below pH 3.0; fα, 0.55→0.52). The difference in pH‐profile of fα between BMA and HSA might be due to the microheterogeneity. The 1H‐NMR spectra and cross‐relaxation times (TIS) from irradiated to observed protein protons, which reflect the structural fluctuation and/or mobilability in proteins, were measured on the N‐, F‐, E‐forms of HSA and BMA, and the N*‐form (8.23 M urea, neutral pD) of iodoacetamide‐blocked HSA (IA‐HSA) and bovine serum albumin (IA‐BSA). The 1H‐NMR spectra and elongations of TIS values for the F‐ and E‐forms of HSA and the E‐form of BMA were quite similar to those for the N*‐form of IA‐HSA and IA‐BSA, indicating the liberation of the intramolecular motion in the F‐ and E‐forms. Those for the F‐form of BMA were intermediate between the N‐ and E‐form. The present results together with the reported data on hydrodynamic radii and D–H exchange reaction, indicate that the F‐form of HSA and presumably BMA has a native‐like globule form with a highly helical state and fluctuating tertiary structure. Thus, all of the present findings on the F‐form of serum albumin seem to be in accord with the structural features for the F‐form suggested by Foster's group (1‐3, 19, 20, 22, 23) and the molten globule state demonstrated by Dolgikh et al. (40), and Ohgushi and Wada (36, 37).

Design of protein homocystamides with enhanced tumor uptake properties for 19F magnetic resonance imaging

“…HSA was dissolved in phosphate buffer with saline (pH 7.4), and concentrations were determined with a UV-1800 spectrometer (Shimadzu, Japan), assuming E (1%, 1 cm) at 280 nm to be 5.30. 72 The SH content of albumin was determined using the Ellman's method as described in the literature 55 at pH 8 and employed DTNB spectrophotometrically at 412 nm (ε = 1.36 × 10 4 M −1 cm −1 ) with a UV-1800 spectrometer (Shimadzu, Japan). It was calculated that the constant absorbance value corresponded to an albumin preparation, which contained 0.67 sulfhydryl group per protein molecule.…”

Comparative 1H NMR studies on the structural looseness of the aged (A) and non-aged (N) bovine mercaptalbumin in the alkaline region

“…The 1 H NMR samples for the N-(pD 5.31), F-(pD 4.00) and Eforms (pD 3.02) of BMA in 0.10 M NaCl were prepared, as described previously [23][24][25]. D 2 O (99.75%, Lot 2919), DCl (Lot 2906) and NaOD (Lot 2911) were purchased from Merck (Darmstadt, Germany).…”

“…Chemical shifts were measured in parts per million (ppm) downfield from an external reference (TSP). Spinecho 1 H NMR spectra were obtained using a [90 • − ( − 180 • − ) n ] spin-echo pulse sequence with = 0.001 s and n = 10 (0.020 s CPMG spectra), as reported previously [17,18,25,26]. Spin-lattice relaxation times (T 1 ) were measured by the inversion recovery method with a pulse sequence of [180 • − − 90 • − acquisition − T] n with T being more than 5 times T 1 .…”

“…For the cross-relaxation time (T IS ) measurements by the inversion recovery method, a built-in decoupling f 2 -channel was used in the gated decoupling mode to provide a pre-saturation rf-pulse (f 2 -irradiation) at the desired frequency of spectra at desired power level during and T in the pulse sequence of the T 1 measurement [20][21][22]. The actual strength of f 2 -power at the sample position, calibrated using BSA gel (BSA * gel; refer footnote 3 ), was chosen to be in the range of 39-107 Hz in H 2 /2 units [17,18,[23][24][25][26]33]. After long-time f 2 -irradiation on the S-spin system, the magnetization of the observed I-spin system will reach an equilibrium value (I ∞ ).…”

“…Compared with the current advanced understanding of the physicochemical characteristics of the N-F (pH 4.4-3.8) and N-B transitions (pH 7.0-9.0), much less is known about the N-A isomerization [1,2]. To study the mechanism of the above mentioned salt effect on k + and k − , we have studied the structural looseness of the N-and A-forms of BMA at neutral and alkaline pD in ∼0 and 0.10 M NaCl using 1 H NMR cross-relaxation times (T IS ) from irradiated to observed protein protons [20][21][22] we reported previously for the N-F [23][24][25] and N-B transitions [17] and the N-A isomerization [18] of BMA and BSA and for the native-molten globule transition of ovalbumin in the acidic region [26].…”