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Structure and Function of Humanc-Reactive Protein
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Alok Agrawal, John M. Kilpatrick, John E. Volanakis
CRP is made up of five identical, noncovalently bound subunits4 exhibiting a planar pentagonal appearance in electron micrographs.2 A molecular weight of 118,000 has been calculated for the native pentameric molecule from sedimentation equilibrium studies.5 The primary structure of CRP has been determined by protein sequencing,6 and has been subsequently confirmed and corrected by sequencing cDNA and genomic clones.7,8 Each subunit of CRP consists of 206 amino acid residues with a calculated molecular weight of 23,017.7,8 A single disulfide bond links the two half-cystines at positions 36 and 97.6 There are no potential N-glycosylation sites in the amino acid sequence and no carbohydrate is present in human CRP.4 Several regions of the amino acid sequence of CRP have been found to be homologous to regions of known function in other proteins, particularly PCh-binding myeloma proteins, calmodulin, tuftsin, and histones (Table 1).
Isolation and Characterization of Pregnancy-Related Proteins
Published in Gábor N. Than, Hans Bohn, Dénes G. Szabó, Advances in Pregnancy-Related Protein Research, 2020
For determining the molecular weight in the ultracentrifuge, the sedimentation equilibrium method was used. The concentration of the protein was adjusted for this purpose to an adsorbance of about 1.0. The determination was affected at 9000 rpm. Registration was carried out with an ultraviolet-absorption lens at 280 nm using a photoelectric scanner. In the tables, the molecular weights are expressed in kilodaltons (kDa).
Ferrihemoglobin in Normal Blood
Published in Manfred Kiese, Methemoglobinemia: A Comprehensive Treatise, 2019
Kuma and Inomata208 achieved a 129,000-fold purification of a NADH-ferrihemoglobin reductase from human red cells by adsorption on DEAE cellulose, ammonium sulfate fractionation, chromatography on Sephadex columns, and starch block electrophoresis. In disc electrophoresis only one yellow band was seen. The enzyme was assumed to have FAD as the prosthetic group. From flavin content the minimal molecular weight was calculated to be 34,400, and gel filtration indicated a molecular weight of 33,000. The purified enzyme reduced ferrihemoglobin very slowly; less purified preparations were a little more active. The authors point out that the existence of an intermediate electron carrier between the flavoprotein and ferrihemoglobin is strongly suggested. Adachi and Okuyama209 purified and crystallized a pyridine nucleotide dehydrogenase from bovine red cells. The enzyme was assayed by its diaphorase activity. It showed a specific activity 14,000 times higher than hemolysate and reacted with NADH three times more rapidly than with NADPH. The reaction with ferrihemoglobin or other hemoproteins was not tested. From sedimentation equilibrium the molecular weight was calculated to be 29,000. The flavin content of the recrystallized enzyme was as low as 0.01 mol of flavin per mol of enzyme.
Binding affinity in drug design: experimental and computational techniques
Published in Expert Opinion on Drug Discovery, 2019
Visvaldas Kairys, Lina Baranauskiene, Migle Kazlauskiene, Daumantas Matulis, Egidijus Kazlauskas
While sedimentation velocity experiments can be used for Kb measurements [39], sedimentation equilibrium is better suited for it. In these experiments, the sample is ultracentrifuged until equilibrium between the centrifugal force and diffusion is reached. The distribution of molecules is tracked throughout experiment measuring absorbance, refraction, or more sensitive fluorescence [40]. This distribution depends solely on the molecular mass and not shape. Kd can thus be calculated based on how the curvature changes at different protein: ligand ratios [41]. Naturally, it can only be applied for interactions with sufficient difference in molecular weight (the precision is usually 1–2%).
Molecular recognition requires dimerization of a VHH antibody
Published in mAbs, 2023
Christopher A. Smith, Gregory J. Sonneson, Robert J. Hoey, Jennifer M. Hinerman, Kimberly Sheehy, Richard Walter, Andrew B. Herr, James R. Horn
To better explore the oligomeric nature of the anti-caffeine VHH, beyond the original size exclusion chromatography analysis,20 AUC sedimentation velocity experiments were pursued. These experiments revealed that at 30 µM anti-caffeine VHH, the protein exists in a monomer-dimer equilibrium, but upon addition of 15 µM caffeine, the equilibrium shifts to favor the dimeric form (Figure 3). Notably, the dimer interface F47R variant is monomeric in solution at 30 µM, suggesting that the Phe to Arg mutation disrupts the dimer interface as designed. Sedimentation equilibrium experiments were performed to verify the oligomerization state of the samples and to determine equilibrium assembly constants (Figure 3). As suggested by the velocity data, all equilibrium data for wild-type VHH were consistent with a monomer-dimer equilibrium in solution that was modulated by the binding of caffeine. The VHH alone exhibited a monomer-dimer KD of 23.5 µM (15.4–35.7 uM), compared to a KD,obs of 848 nM (414 nM−1.6 uM) in the presence of a stoichiometric amount of caffeine (values in parenthesis represent 95% confidence limits). Thus, caffeine binding resulted in a nearly 28-fold higher observed dimerization affinity (ΔΔG°dim = −1.9 kcal/mol). Equilibrium data revealed that the F47R-VHH variant sedimented as a monomer even in the presence of 15 µM caffeine, indicating that this point mutation dramatically destabilizes the dimeric assembly. Given the velocity data indicating that F47R-VHH was completely monomeric in the absence of caffeine, this sample was not further tested by sedimentation equilibrium experiments. Notably, the ITC data for variant F47R (as well as Y[100B]R) could only be reasonably fit to a binding model when fixing the stoichiometry to a 2:1 and not 1:1 interaction, which agrees with AUC data that dimerization is necessary for caffeine recognition. These data support a linked equilibria model where VHH dimerization occurs first, followed by a second caffeine-binding event (Figure 4A).