Circular dichroic and fluoropolarimetric studies on tryptophyl residues in acid-induced isomerization of bovine plasma albumin*

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring circular dichroic spectra and fluorescence polarization of tryptophyl residues. Decreases in the magnitude of ellipticities at 208, 222, 262 and 268nm were observed in the N-F transition and acid-expansion. However, increases in the magnitude of ellipticities at 295–300nm observed in the initial part of the N-F transition exactly correlated with the increase of rotational relaxation time of tryptophyl side chains obtained by fluorescence polarization measurement.     

CD-resolved secondary structure of bovine plasma albumin in acid-induced isomerization*

Bovine plasma albumin (BPA) showed the acid-induced two-step transition, the N-F transition and acid-expansion. Changes in fractions of α-helix (f_α), β-form (f_β) and unordered form (f_R) in the acid-induced isomerization of BPA were studied using the method of Chen et al. (1972) with two constraints: f_i = 1, 0 ± f_i ± 1. pH-profiles of f_α and f_R showed the two-step change, one corresponding to the N-F transition and the other to the acid-expansion in 0.10 m KCl and in 0.02 m NaClO₄. pH-profile of f_β showed one-step change, correlating to the later part (lower pH side) of the N-F transition. The N-F transition might thus involve the helix ± β and helix ± coil transitions.     

Water structure in modified bovine plasma albumin (BPA*) gel and bovine mercaptalbumin solution† 1 H-n.m.r. studies

Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 8%, though proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F-form of BMA in the solution state and of BPA* in the gel state were studied by measuring ¹ H-n.m.r. spectra, spin-lattice relaxation time (T₁) and cross relaxation time (T_IS) between irradiated and observed protons. Protein concentration-dependent changes of T₁ of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F-form of BMA in the solution state. T_IS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.     

1H-n.m.r. studies on cross-relaxation phenomena in bovine mercaptalbumin (BMA) solution and partially hydrolyzed bovine plasma albumin (BPA*) gel

Bovine plasma albumin Fr. V(BPA) contains small amounts of proteolytic enzyme which catalyzes a very limited cleavage of BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA(BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 7%. Highly purified proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12.4%. after 5 days incubation at 35°. Cross-relaxation times (T_IS) between irradiated protein protons and observed protons, such as side chain and water protons, were studied on BMA solution and BPA*-gel. T_IS values of BMA solution, obtained by the saturation transfer (SATUR) and inversion recovery (INVER) methods, were a single kind of T_IS for each side chain. Those of BPA*-gel by the SATUR method indicated the presence of two kinds of T_IS, that is, short and long T_IS values for each side chain. However, those by the INVER method showed a single kind of T_IS for each side chain, corresponding to the long T_IS value by the SATUR method. The short T_IS values of BPA*-gel, observed by the SATUR method, may be due to immobile joint parts of fibrous BPA* aggregates. T_IS values from protein to water protons (T_IS(HDO)) in BPA*-gel, obtained by the INVER method, were far shorter than those in BMA solution, indicating a large amount of hydration of BPA* and rapid exchange between bound and bulk water in the gel state.     

Circular dichroic and 1 H-NMR studies on the aged form of bovine plasma albumin

Bovine plasma albumin (BPA) has 17 disulfide bonds and approximately one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). 1) Fractions of α-helix (f_α) and β-form (f_β) of iodoacetamide-blocked non-aged BPA (IA-BPA) at pH 6.5 (the N-form) and 9.0 (the B-form) in the absence of added salt were 0.70, 0.12 and 0.62, 0.18, respectively (Era et al. (1990)). However, there were no changes in f_α and f_β of the iodoacetamide-blocked A-form (IA-A-form) over the pH range from 5.5 to 9.1 in the absence of added salt or in 0.10 m KCl(f_α～ 0.60, f_β～ 0.20), indicating that the secondary structure of the IA-A-form might be similar to that of non-aged IA-BPA at pH 9.0 (B-form) in the absence of added salt, that is, the frozen B-form, stabilized covalently by the repairing of disulfide bonds. 2) The rigidity of the A- and IA-A-forms, as monitored by cross-relaxation times between irradiated and observed protein protons, was similar to or slightly higher than that of non-aged IA-BPA or BMA. However, spin-echo ¹H-NMR spectra indicated that side chains of the IA-A-form, such as ?-CH₂ of Lys, -CH₃, in the absence of added salt are more mobile than those of non-aged IA-BPA. 3) The ratio of the hydrodynamic volume of the A-form to that of non-aged BPA at pH5.5 was approximately 1.1.     

Investigation of the 35–62 conserved helix and disulfide loop of bovine prothrombin using an anti-peptide antibody

A 28-residue peptide corresponding to the 35–62 region of bovine prothrombin fragment 1 (BF1) was synthesized by solid-phase methods. In BF1 this region consists of three conserved aromatic residues within an α-helical region followed by a disulfide loop. This synthetic peptide was used to produce murine monoclonal antibodies (MAbs) that would recognize and bind native BFI. Antibody AH.Ab.E3, an IgGl antibody that was isolated and cloned, recognized and bound to both the synthetic peptide and the BF1 molecule. Residues 55–59 (REKLN) were shown to be critical for antibody binding. This MAb was subsequently used to study the 48-62 disulfide loop region of BFl. MAb AH.Ab.E3, which has been shown to bind the BF1 calcium-dependent conformation (BFl:Ca), does not appear to perturb the binding interaction between BFI:Ca and phospholipid (PL) vesicles as studied by light scattering methods. © Munksgaard 1996.     

Quantitative determination of zidovudine diaryl phosphate triester pro-drugs in rat plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A rapid, simple, and sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS) method was developed and validated for quantitative analysis of 3′-azido-3′-deoxythymidine (zidovudine, AZT) diaryl phosphate triester pro-drugs, in rat plasma using 2′,3′-dideoxy-2′3′-didehydrothymidine (d4T) as internal standard (IS). The analytes were extracted from rat plasma with methanol after protein precipitation. The compounds were separated by HPLC with gradient elution (on a Shim-pack VP-ODS C₁₈ analytical column using a mobile phase of methanol/10 mM ammonium acetate). All the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQs were 10 ng mL⁻¹ for M1, M2, M3, M4, and M5, respectively. Correlation coefficient (r) values for the linear range of 10–10,000 ng mL⁻¹ were greater than 0.999 for all the analytes. The intra-day and inter-day precision and accuracy were higher than 7.13%. The relative and absolute recovery was above 72% and no matrix effects were observed for all the analytes. This validated method provides a modern, rapid, and robust procedure for the pharmacokinetic studies of the pro-drugs after intravenous administration to rats. Some important results of AZT diaryl phosphate triester pro-drugs concerning chemical effect on pharmacokinetic performance are also studied.     

Extremely fast hydrogen exchange of ribonuclease-(1–118) as compared with native RNase A and its implication for the conformational energy state

RNase-(1–118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the ‘protected’ amide protons of RNase-(1–118) over RNase A. A calculation shows a large increase in the rate of ‘opening’ of the structure. The exchange rate of the protected amide protons of RNase-(1–120) is slower than RNase-(1–118) but much faster than RNase A. Binding with a synthetic complementing fragment (114–124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1–118). It has previously been shown that binding with a complementing fragment of RNase-(1–118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.