Structure-Function Relationships of Folded and Unfolded Amaranth Proteins

In the present study intrinsic fluorescence (IF), surface hydrophobicity (S₀), electrophoresis, circular dichroism (CD), differential scanning calorimetry (DSC), and computer-generated analysis were used to study folded and unfolded proteins from Amaranthus hypochondriacus (A.h.). Globulin (Glo), albumin (Alb), and albumin subfractions (Alb-1 and Alb-2) extracted from A.h. were denatured with urea and guanidine hydrochloride (GdnHCl). The obtained results provided evidence for differences in their secondary and tertiary structures. The most stable was Glo, followed by Alb-2 fraction. The larger percent of denaturation in protein fractions, which is associated with enthalpy and the number of ruptured hydrogen bonds, corresponds to the disappearance of ahelix. Predicted functional changes in model protein systems in response to processing conditions may be encountered in the pharmaceutical and food industries. Our very recent studies have shown that the sustained drug release rate from ovalbumin matrix depends on the conformational state of the protein (Gorinstein et al., 1995; Zemser et al., 1994). Physicochemical characterization of the structural stability of some plant globulins was observed (Gorinstein et al., 1996). The function-structure relationships of amaranth protein fractions were not investigated in spite of their high nutritional value. These proteins can be used as potential matrices for drug release and food ingredients. Therefore in this report the function-structure relationships of albumin and globuliu fractions from amaranth have been studied. Alb and Glo fractions were extracted from defatted meal of A.h. with 0.5 M NaCl, then separated by dialysis against water. Alb-1 was extracted with 0.5 M NaCl or water used as the first solvent. Another fraction (Alb-2) was extracted with water after removing Alb-1 and Glo. Then all fractions Glo, Alb, Alb-1, and Alb-2 were dialyzed and freeze-dried (Gorinstein et al, 1996; Konishi et al, 1991). IF measurements of Glo, Alb, Alb-1, and Alb-2 at excitation wavelengths (nm) of 274 and 295 (Model FP-770 Jasco spectrofluorometer) were carried out and fluorescence intensity / was measured. Molecular weight was determined by sodium polyacrylamide gel electrophoresis (SDS-PAGE). S₀ was determined by l -anilino-8-naphthalenesulfonate (ANS) -fluorescent probe measurements (Akitá and Nakai, 1990). The thermal denaturation was assessed with a Perkin Elmer DSC System. All thermodynamic parameters were found. CD spectra in the presence of GdnHCl were recorded on a Jasco J-600 spectropolarimeter. Secondary structure content was calculated using a Provencher nonliner leastsquares curve-fitting program and the results of CD measurements (Gorinstein et al., 1996). Two dimensional gel electrophoresis (2DE), blotting and sequence of amino acids, and search for homology based on computer-generated data of the sequence of amino acids residues were used for a peptide plot (Kyte and Doolittle, 1982). 2DE of Glo showed protein spots in the range of 17, 22, 28, and 30-38 kDa. Glo and Alb-2 contained similar major 2DE patterns in the range of 36 -38 kDa. The main spots of 17, 22, and 28 kDa were sequenced: spot 1 (17 kDa); EDIVPSG(T); spot 2 (22 kDa): KKKNKPFNFFKEDPD; spot 3 (28 kDa): KSKDKKKNDDPYY. Peptide sequences of these three spots were compared. Homology was found with other plants. Fluorescence spectra of Glo, Alb, Alb-1, and Alb2 demonstrated peaks (nm) at 338, 346, 346, and 351, respectively. This means that tryptophan residues are situated closed to the surface of the molecule in the case of albumins (Alb, Alb-1, and Alb-2) and is consistent with less compact and more hydrophobic structure in comparison with Glo. At γ_excitation = 274 nm very slight shoulders were seen only in Glo γ_emission = 308 nm; I = 0.188) and in Alb-2 (7_emission = 308.5 nm, I = 0.206), which was evidence of tyrosine. At 7excitation = 295 nm tyrosine was not shown. Fluorescence measurements showed a decrease in fluorescence intensity and a shift in the maximum of emission reflecting unfolding of these proteins with urea (Table 1). The percentage of denaturation for Glo, Alb, Alb-1, and Alb-2 proteins with 8 M urea was 28.6, 55.2, 52.2, and 33.3%, respectively. The fluorescence intensity gradually decreased with the increase in urea concentration The difference in the extent of denaturation between the protein fractions may be explained by the differences in amounts of amino acids and by the sulfur bridges existing in such proteins. Surface hydrophobicity was the highest for Alb. Alb-2 and Alb-1 had lower values than Glo. Glo has more compact tertiary structure and less hydrophobic surface than albumin. Increase in surface hydrophobicity was correlated with the increase in the extent of protein denaturation and can be explained by the altered, par-tially unfolded globulin and albumin conformation induced by urea. Unfolding of albumins and globulin is a result of promoted interactions between exposed functional groups which involve transconformations of αhelix, % (in native: Glo = 31; Alb = 5; Alb-1 = 4, and Alb-2 = 16), β-sheet, % (in native: Glo = 27; Alb = 37; Alb-1 =41, and Alb-2 = 26), and aperiodic structure, % (native: Glo = 42; Alb = 58; Alb-1 = 55, and Alb-2 = 53). Denaturation brought an increase in β-sheet content, % (Glo = 47; Alb = 53; Alb-1 = 57, and Alb-2 = 60) reduction of ot-helix, % (Glo = 11 ; Alb = 0; Alb-1 = 0, and Alb-2 = 8). The thermal denaturation of albumins and globulin expressed in terms of temperature of denaturation (T_a, °C), enthalpy (ΔH, kcal/mol), and entropy (AS, kcal/mol K) showed similar changes in protein fractions as were characterized by fluorescence. Number of hydrogen bonds n ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). These results show that during denaturation 19 hydrogen bonds were ruptured in native Glo compared with 29 in Alb and 21 in Alb-2. Predicted hydrophobicity, the position in the sequence of basic and hydrophobic, and of acidic and hydrophilic amino acids (Kyte and Doolittle plots of hydropathy), and changes in α-helix and β-sheet were calculated. In summary, thermal denaturation parameters, S₀, and the content of secondary structure show that Glo and Alb-2 have similar conformational changes and degree of denaturation, suggesting that Alb-2 is also a storage protein like Glo.