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1.
Light-scattering and related studies on the polymerization behavior of the protein from the PM2 strain of TMV show that in phosphate buffer of ionic strength 0.1, the maximum extent of temperature-mediated polymerization occurs at pH values lower than in the case of TMV protein. The pH range of temperature-induced polymerization is from 5.0 to 6.0, contrasted with 5.0 to 7.5 for TMV protein. Velocity sedimentation studies show that PM2 protein at room temperature in phosphate buffer (I = 0.1) has sedimentation coefficients of 174 S, 104 S, and 4.3 S at pH values of 4.89, 5.53, and 7.5. Electron microscope studies show that at room temperature in phosphate buffer of 0.1 ionic strength at pH 5.53, PM2 protein has structures resembling essentially that of stacked double discs with an occasional helical structure. Similar studies of PM2 protein in 0.1 M ammonium acetate buffer at pH 5.2 show single, double, and double-double helices. 相似文献
2.
The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein. 相似文献
3.
Bolton GR Boesch AW Basha J Lacasse DP Kelley BD Acharya H 《Biotechnology progress》2011,27(1):140-152
Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein-ion binding. Data for the pH offsets of an Fc-fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc-fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect. 相似文献
4.
Light-scattering and related studies on protein of Dahlmense strain of tobacco mosaic virus (DTMV) show that its polymerization characteristics are considerably different from those of TMV protein. At pH 6.0 in phosphate buffer (I = 0.1), the extent of polymerization of DTMV protein is greater than that of TMV protein, they are nearly the same at pH 6.25, and that of DTMV protein is less than that of TMV protein at pH 6.5. At pH 7.0 and 7.5, DTMV protein polymerizes more readily than TMV protein. Similar studies in phosphate buffer (I = 0.05) show that the extent of polymerization for DTMV protein is less than that of TMV protein at pH 6.0 and almost negligible at pH 6.25. Acid-base titration studies show that, upon temperature-mediated polymerization, about 2 H+ ions are bound per monomer of DTMV protein at pH 6.O.Electron microscope studies show that DTMV protein exists at room temperature as double discs and polymerized rods in phosphate buffer at pH 7.5, I = 0.1; at pH values below 6.5, DTMV protein is entirely in the form of polymerized rods. Velocity sedimentation studies of DTMV protein at room temperature are in agreement with these findings. At low temperatures, except at pH 7.5, most of the material sedimented with an s value of around 25 S. Thus, at low temperatures, except at pH 7.5, DTMV protein in solution is in the form of particles the size of double discs with an of 596,000 g/mole or even larger. Therefore, temperature-mediated polymerization of DTMV protein at pH values below 6.5 in phosphate buffer (I = 0.1) and below 6.25 in phosphate buffer (I = 0.05) involves particles at least as large as double discs as the starting material. 相似文献
5.
Youngbin Baek Deyu Yang Nripen Singh Abhiram Arunkumar Sanchayita Ghose Zheng Jian Li Andrew L. Zydney 《Biotechnology progress》2017,33(6):1555-1560
Diafiltration is used for final formulation of essentially all biotherapeutics. Several studies have demonstrated that buffer/excipient concentrations in the final diafiltered product can be different than that in the diafiltration buffer due to interactions between buffer species and the protein product. However, recent work in our lab has shown variations in solution pH that are largely independent of the protein concentration during the first few diavolumes. Our hypothesis is that these pH variations are due to nonidealities in the acid‐base equilibrium coefficient. A model was developed for the diafiltration process accounting for the ionic strength dependence of the pKa. Experimental results obtained using phosphate and histidine buffers were in excellent agreement with model predictions. A decrease in ionic strength leads to an increase in the pKa for the phosphate buffer, causing a shift in the solution pH, even under conditions where the initial feed and the diafiltration buffer are at the same pH. This effect could be eliminated by matching the ionic strength of the feed and diafiltration buffer. The experimental data and model provide new insights into the factors controlling the pH profile during diafiltration processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1555–1560, 2017 相似文献
6.
An extracellular aminopeptidase, purified 465-fold from culture filtrates of Bacillus licheniformis, was found to be a metalloenzyme consisting of a single peptide chain. Sedimentation equilibrium yielded a molecular weight of 43,270 and two polyacrylamide electrophoretic procedures gave values of 37,500 and 36,000, respectively. The activity of the enzyme was inhibited severely by 1,10-phenanthroline and to a lesser extent by EDTA, cyanide, and fluoride. The addition of Co2+ ions greatly stimulated enzymatic activity, but analysis of the purified enzyme revealed the presence of zinc, not cobalt, in stoichiometric quantities. Moreover, the ratio of zinc to protein was found to increase during fractionation, reaching a final value corresponding to 1 g-atom/mol. The aminopeptidase possessed characteristics of a euglobulin, sparingly soluble in water and dilute buffer solutions, but soluble in buffers containing higher concentrations of salts. Both activity and pH optimum were substantially influenced by ionic strength; as the latter was increased over the range from 0.01 to 0.1, activity increased and the pH optimum was shifted to more acidic values. Enzymatic activity was affected by the identity of the buffer, being markedly greater in Tris-HCl than in sodium barbital and strongly inhibited by phosphate. The Bacillus aminopeptidase hydrolyzed substrates with unsubstituted amino groups of the l configuration, including dipeptides, aminoacylnaphthylamides, and amino acid amides. 相似文献
7.
Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA. 相似文献
8.
Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated. 相似文献
9.
Purified rat ceruloplasmin is extraordinarily unstable in storage at –70 °C. In a 20 mM phosphate buffer, pH 7.0, the ferroxidase and amine oxidase of ceruloplasmin are over 90% inactivated within two weeks. Holoceruloplasmin stored for three months in a 20 mM barbital buffer (or acetate buffer), pH 7.0 (or pH 5.5) was transformed into an apo-protein and amine (o-dianisidine) oxidase of ceruloplasmin was inactivated by 50–55%. The patterns of ferroxidase activity loss were similar to those of amine oxidase activity loss. On the contrary, when holoceruloplasmin was mixed with rat serum albumin, transformation into apoceruloplasmin was significantly prevented in a 20 mM barbital buffer, pH 7.0 (or 20 mM acetate buffer, pH 5.5). Consequently, ferroxidase and amine oxidase activities of ceruloplasmin were not inactivated and the immunochemical reactivity was not changed. These results can be applied for laboratorial and clinical purposes. 相似文献
10.
Rafikova ER Kurganov BI Arutyunyan AM Kust SV Drachev VA Dobrov EN 《The international journal of biochemistry & cell biology》2003,35(10):1452-1460
To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein. 相似文献
11.
Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system 总被引:1,自引:0,他引:1
Y Seo M Murakami H Watari Y Imai K Yoshizaki H Nishikawa T Morimoto 《Journal of biochemistry》1983,94(3):729-734
To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C. 相似文献
12.
Seidegård J Grönquist L Tuvesson H Gunnarsson PO 《In vitro cellular & developmental biology. Animal》2009,45(1-2):32-34
The stability of two nitrosoureas, tauromustine and lomustine, has been investigated in different media and buffers. All media tested, except Leibovitz's L-15 medium, significantly increased the degradation rate of the investigated nitrosoureas at pH 7.4. Sodium bicarbonate seems to be the cause of the observed increase of the degradation rate, since it provides the main buffering capacity of all the media except for Leibovitz's L-15 medium, which is based on phosphate buffer. Other ingredients in the media, such as amino acids, vitamins, and inorganic salts, or the ionic strength of a buffer, did not have any major effect on the degradation rate of the nitrosoureas. These results suggest that media containing carbonated buffer should be avoided when the anti-tumor effect of nitrosoureas is to be investigated in different cell cultures. 相似文献
13.
The solubility of cellulase extracted from the abscission zones of citrus leaf explants (Citrus sinensis L. Osbeck) in sodium phosphate buffer depends on the pH of the extracting solution and, to a lesser extent, on the ionic strength. By increasing molarity from 0.01 to 0.16, the solubility of cellulase increased from 51% to 89% at pH 6.1 and from 70% to 98% at pH 7. In all cases, residual cellulase was further extracted from the pellet by buffer containing 1 m NaCl. Most of the enzymic activity was found in tissues proximal to the separation line, and activity of the cellulase which was soluble in phosphate buffer was closely correlated with abscission at both pH values. When extraction of cellulase at pH 6.1 with phosphate buffer was followed by a reextraction of the pellet with buffer containing 1 m NaCl, the activity of the cellulase soluble in the fortified buffer was also correlated with abscission. Pretreatment of explants with ethylene increased the solubility of cellulase in the phosphate buffer regardless of the pH used at the first extraction. 相似文献
14.
The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes. 相似文献
15.
粘虫中肠α-淀粉酶活性的敏感性研究 总被引:2,自引:1,他引:1
研究了不同酶反应缓冲体系、pH值、氯离子浓度以及噁唑哒嗪对5龄2日粘虫 Pseudaletia separata Walker 中肠α-淀粉酶活性的影响。结果表明,乙酸-乙酸钠缓冲体系(pH 5.8)和磷酸氢二钠-磷酸二氢钠缓冲体系(pH 8.0)有利于增强α-淀粉酶活性,比活力最高分别达到4.49和4.97。在乙酸-乙酸钠缓冲体系(pH 5.8)中,5、10、20、40和80 mmol/L氯离子浓度引起α-淀粉酶活性呈现先减弱后增强的变化规律,而在磷酸氢二钠-磷酸二氢钠缓冲体系(pH 8.0)中仅呈现减弱的趋势。1.4 mmol/L噁唑哒嗪对α-淀粉酶活性的抑制率可达70%,但抑制程度随着反应体系中蛋白含量的增加而逐渐降低。 相似文献
16.
Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.
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We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes. 相似文献
17.
Human lactotransferrin binds 2 Fe3+ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA.The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe3+ from human lactotransferrin are removed stimultaneously.Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed.Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe3+ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe3+ from the ‘acid stable’ site is obtained at pH 4.The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with 59Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site. 相似文献
18.
Joseph A. Laszlo Gary M. Baker Richard A. Dilley 《Journal of bioenergetics and biomembranes》1984,16(1):37-51
Isolated spinach thylakoids retain a slowly equilibrating pool of protons in the dark which are predominantly bound to buffering groups, probably amines, with low pKa values. We have measured the effects of permeant buffers, salts, sucrose, and uncouplers on the retention of the proton pool. Acetic anhydride, which reacts with neutral primary amine groups, was used to determine the protonation state of the amine buffering groups. It was previously shown by Bakeret al. that the extent of inhibition of photosystem II water-oxidizing capacity by acetic anhydride and the increase in derivatization by the anhydride are proportional to, and dependent on, the deprotonated state of the amine buffering pool. Therefore, acetic anhydride inhibition of water oxidation activity may be used as a measure of the protonation state of the amine buffering pool. By this method it is inferred that protons, in a metastable state, were retained by membranes suspended in high pH buffer for several hours in the dark. When both the internal and external aqueous phases were equilibrated with pH 8.8 buffer, the proton pool was released only upon addition of a protonophore. The osmotic strength of the suspension buffer affected uncoupler-induced proton release while ionic strength had little influence. The acetic anhydride-sensitive buffering group(s) of the water-oxidizing apparatus had an apparent pKa of 7.8. We conclude that an array of protein buffering groups reside either within the membrane matrix, or in proteins at the membrane surface, not in equilibrium with the bulk aqueous phases, and is responsible for the retention of the proton pool in dark maintained chloroplasts. 相似文献
19.
Liliana Gianfreda Gennaro Marino Rosaria Palescandolo Vincenzo Scardi 《The Biochemical journal》1974,137(2):199-203
1. The effect of pH change on the reconstitution of aspartate aminotransferase (EC 2.6.1.1), i.e. the reactivation of the apoenzyme with coenzyme (pyridoxal phosphate and pyridoxamine phosphate), was studied in the pH range 4.2-8.9 by using three buffer systems at concentrations ranging from 0.025 to 0.1m. 2. Although the profile of the reconstitution rate-pH curve in the range pH5.2-6.8 (covered by sodium cacodylate-HCl buffer) reflects the influence of the H(+) concentration on the reconstitution process, the profile of the curve in the pH ranges 4.2-5.6 and 7.2-8.25 (covered respectively by sodium acetate-acetic acid and Tris-HCl buffers) appears to be influenced by the ionic strength of the buffer. 3. The reconstitution is also influenced by univalent inorganic ions such as halide ions and, to a lesser extent, alkali metal ions, which are known to alter the water structure. 相似文献
20.
P G Righetti M Fazio C Tonani E Gianazza F C Celentano 《Journal of biochemical and biophysical methods》1988,16(2-3):129-140
The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures. 相似文献