Kinetic Analysis of Enzyme Inactivation under Second-Order Conditions by Use of Substrate-to-Product Progress Curves: Application to the Inhibition of Trypsin by α-1 Proteinase Inhibitor

Özer, İnci

doi:10.1006/abio.1998.2855

Cited by 4 publications

(2 citation statements)

References 16 publications

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“…osmolyte on the activity and stability of trypsin itself, an assay using a fluorescent trypsin substrate (BAAMC) was done in the presence and absence of osmolyte. The assay conditions and BAAMC storage were as described previously (26). The BAAMC cleavage reaction was monitored in an SPEX fluorometer with excitation at 333 nm (slit width, 0.7 nm) and emission at 440 nm (slit width, 10 nm).…”

Section: Methodsmentioning

confidence: 99%

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Ratnaparkhi¹,

Varadarajan²

2001

Journal of Biological Chemistry

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Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein⅐peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 M sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4°C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 M sarcosine increases with temperature, ranging from ؊0.52 kcal mol ؊1 at 20°C to ؊5.4 kcal mol ؊1 at 60°C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.Osmolytes are molecules used in nature to protect organisms against stresses of high osmotic pressure. These compounds have also been found to stabilize the native state of proteins relative to the unfolded state. The mechanism of this stabilization is not completely understood (1-4), although it is believed to result primarily from an unfavorable free energy of interaction between the osmolyte and the unfolded state of the protein (5, 6). Proteins retain activity in the presence of osmolytes suggesting that native state structure and dynamics are not greatly perturbed. However, there is little high resolution structural information available for proteins in the presence of osmolytes. The main classes of osmolytes are sugars, methyl ammonium derivatives, polyhydric alcohols, and amino acids and their derivatives (1). Molar concentrations of all the above classes of molecules have been shown to stabilize proteins. Many organisms accumulate osmolytes under conditions of water stress, such as high salinity, desiccation or freezing. In vivo, amino acid derivatives also counteract the accumulation of urea, which is capable of denaturing proteins. Marine cartilaginous fishes use, as osmolytes, a combination of urea and methylamines, i.e. a denaturant and a stabilizer, in a 2:1 ratio (1,7,8). Stability studies on RNase T1, RNase A, and other proteins have shown that, if the 2:1 ratio of urea:methylamine is maintained, the methylamine is able to counteract the destabilizing effect of the denaturant (1,7,8).In an effort to further our understanding of the basis of osmolyte stabilization of proteins, we have studied the stabilization of the fragment complementation system ribonuclease S (RNase S) 1 in four structurally similar osmolytes, namely sarcosine, betaine, trimethylamine-N-oxide (TMAO), and taurine. RNase S is a complex of two fragm...

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Section: Methodsmentioning

confidence: 99%

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Ratnaparkhi¹,

Varadarajan²

2001

Journal of Biological Chemistry

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“…It has also been used as a trypsin inhibitor in some previous studies (Xiang et al 2006;Wang et al 2008bWang et al , 2009) because it contains a-2-macroglobulin, a large plasma protein (60 to 80kDa) (Dangott and Cunningham 1982) capable of inactivating different types of proteinases (Barrett and Starkey 1973). In addition, another protein in FBS, a-1 antitrypsin, which has a mass of 52 kDa, was reportedly able to specifically and irreversibly inactivate the enzyme trypsin in vitro (Hercz 1986;Ozer 1998). In the FBStreated cartilage tissues, we believed that both of these two kinds of proteins inactivated the trypsin digestion by competing with PGs for binding to trypsin, whereas the delay of inhibition effect by FBS was probably a result of the molecular size of these trypsin-inhibiting proteins.…”

Section: Discussion and Summarymentioning

confidence: 95%

Real-Time Electro-Mechano-Acoustic Imaging for Monitoring Interactions Between Trypsin and Different Inhibitors in Articular Cartilage

Zheng

Wang

Butt

2011

Ultrasound in Medicine & Biology

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A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

Tacal

Özer

2002

Journal of Biochemical and Biophysical Methods

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Kinetic Analysis of Enzyme Inactivation under Second-Order Conditions by Use of Substrate-to-Product Progress Curves: Application to the Inhibition of Trypsin by α-1 Proteinase Inhibitor

Cited by 4 publications

References 16 publications

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Real-Time Electro-Mechano-Acoustic Imaging for Monitoring Interactions Between Trypsin and Different Inhibitors in Articular Cartilage

A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

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