We have analyzed the adsorption of protein to the surfaces of silica nanoparticles with diameters of 6, 9, and 15 nm. The effects upon adsorption on variants of human carbonic anhydrase with differing conformational stabilities have been monitored using methods that give complementary information, i.e., circular dichroism (CD), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel permeation chromatography. Human carbonic anhydrase I (HCAI), which is the most stable of the protein variants, establishes a dynamic equilibrium between bound and unbound protein following mixture with silica particles. Gel permeation and AUC experiments indicate that the residence time of HCAI is on the order of approximately 10 min and slowly increases with time, which allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. The effects on the protein conformation from the interaction have been characterized using CD and NMR measurements. This study shows that differences in particle curvature strongly influence the amount of the protein's secondary structure that is perturbed. Particles with a longer diameter allow formation of larger particle-protein interaction surfaces and cause larger perturbations of the protein's secondary structure upon interaction. In contrast, the effects on the tertiary structure seem to be independent of the particles' curvature.

1. The steady-state kinetics of the interconversion of CO, and HCO; catalyzed by human carbonic anhydrase C was studied using 'H,O and ' H 2 0 as solvents. The pH-independent parts of the parameters k,,, and K , are 3 -4 times larger in 'H,O than in ,H,O for both directions of the reaction, while the ratios k,,,/K, show much smaller isotope effects. With either C 0 2 or HCO; as substrate the major pH dependence is observed in k,,,, while K , appears independent of pH. The pK, value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2 H 2 0 than in ' H 2 0 .2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhydrase C is approximately 35 faster in 'H20 than in ' H 2 0 . In both solvents the pK, values of the pH-rate profiles are similar to those observed for the C0,-HCO; interconversion.3. It is tentatively proposed that the rate-limiting step at saturating concentrations of C 0 2 or HCO; is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO, and HCO; involves a proton transfer or not.Carbonic anhydrase is a highly efficient catalyst of the reversible interconversion of C 0 2 and HCO,. In a buffered solution not far from neutrality, where Cog-as well as free H + and OH-can be neglected, the stoichiometry of the reaction is CO, + H,O + B e HCO; + BH+, where B and BH' are the basic and acidic buffer components, respectively. Regardless of the specific reaction mechanism, the hydration of C 0 2 must be coupled to the splitting of water, formally into H + and OH-. At some stage of the reaction the OH-ion becomes integrated with C02, while the H + ion ultimately combines with the buffer base. In the reverse reaction OH ~ derived from HCO; must combine with H', originating from the buffer acid, to form H,O. Thus, proton transfers are compulsory ingredients in any mechanism of this reaction.Because of the extremely rapid turnover observed for the enzyme-catalyzed reaction, lo5-lo6 s-' at 25 that H,CO, should be regarded as the substrate species specifically combining with the active site. In effect this means that H + is transported bound to HCO;. It follows from this model that additional proton transfers would have to occur within the enzyme-substrate complex, for example in a concerted reaction as proposed by Kaiser and Lo [3].Arguments against H2C03 as the substrate species combining with the active site have been given by several authors [4-61 pointing out that this would require a second-order rate constant for the binding step exceeding those of diffusion-controlled reactions measured in simpler systems. Alternatively it was suggested that H + is transported between solvent and active site by buffer components acting as proton donors and acceptors. In this case no second-order rate constant involved in the catalytic cycle would have to be greater than lo8-lo9 M-' s -l , and it is not necessary to invoke novel phenomena such as surface diffusion [2,7] to rationalize the e...

The circular dichroism (CD) spectrum of human carbonic anhydrase II (HCAII) has been investigated using various mutants of the enzyme in which tryptophans have been replaced by site-directed mutagenesis. HCAII contains seven tryptophans which are believed to significantly contribute to the CD spectrum in both the near- and far-UV regions. By substituting the tryptophans one at a time, the spectral effects of the individual tryptophans were studied. The near-UV spectrum of HCAII is very complex, with multiple Cotton effects. This complexity has been attributed to aromatic amino acids, especially tryptophans, located in asymmetric aromatic clusters in the molecule. CD spectra of the individual tryptophans were calculated as difference spectra between the CD spectrum of HCAII and those of the tryptophan mutants. These spectra showed that the tryptophans contributed to the CD spectrum in almost the entire wavelength region investigated (180-310 nm). Summation of the individual tryptophan CD spectra in the near-UV region yielded a spectrum that was qualitatively very similar to that of HCAII, showing that the tryptophans are the major determinant for this part of the CD spectrum. Since tryptophans were also demonstrated to contribute significantly in the far-UV region, tryptophans can interfere considerably with the assignment of changes in CD bands to changes in secondary structure content during folding reactions. Moreover, because of this substantial interference, predictions of the amount of various types of secondary structure from CD data from the far-UV region are made more difficult. These findings are probably of general importance for proteins that, like HCAII, contain several tryptophans.(ABSTRACT TRUNCATED AT 250 WORDS)

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.