Photooxidation of crystalline chymotrypsin in the presence of methylene blue

Weil, L.; James, Sarah; Buchert, A.R.

doi:10.1016/0003-9861(53)90200-8

Cited by 182 publications

(38 citation statements)

References 25 publications

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“…I n these and subsequent papers Jansen and Balls with co-workers, as well as other authors (see [3]), showed that the phosphorylation of a-chymotrypsin or trypsin is accompanied by a simultaneous decrease in the esterase, amidase and peptidase activities. The fact of the existence of the common active site in a-chymotrypsin was also supported by the data on photooxidation of the enzyme [4] in which a-chymotrypsin inactivates with respect to both esteric and amide substrates. However, these data only suggest that a-chymotryptic and tryptic hydrolyses of amide and esteric substrates occur on the same site of the enzyme molecule.…”

supporting

confidence: 56%

“…I n these and subsequent papers Jansen and Balls with co-workers, as well as other authors (see [3]), showed that the phosphorylation of a-chymotrypsin or trypsin is accompanied by a simultaneous decrease in the esterase, amidase and peptidase activities. The fact of the existence of the common active site in a-chymotrypsin was also supported by the data on photooxidation of the enzyme [4] Abbreviations. Z-Arg-Nan, N-carboxbenzoxy-L-arginine m-nitroanilide ; Bz-Arg-Nan, N-benzoyl-DL-arginine p-nitroanilide; Bz-Arg-OEt, N-benzoyl-L-arginine ethyl ester.…”

supporting

confidence: 54%

See 1 more Smart Citation

The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

Березин¹,

Kazanskaya²,

Klyosov³

et al. 1973

European Journal of Biochemistry

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The applicability of the acylenzyme mechanism to the tryptic hydrolysis of the amide bond has been studied. It was found that trypsin acetylated a t the hydroxyl group of the "active" serine or acetyl (Ser-183)trypsin displays no activity with respect to both esters (N-benzoyl-L-arginine ethyl ester and p-nitrophenyl-p'-guanidinobenzoate) and anilides (N-carbobenzoxy-L-arginine m-nitroanilide and N-benzoyl-DL-arginine p-nitroanilide). It was also found, that deacetylation of acetyl(Ser-183)trypsin is effectively promoted by N-carbobenzoxy-L-arginine m-nitroanilide (Z-Arg-Nan) taken a t a high concentration. As was found by means of a detailed kinetic analysis of experimental results, the binding of this anilide with acetyl(Ser-183)trypsin increases the rate constant of deacylation of the latter by as many as 23 times. This effect which results in a rapid increase in the concentration of native trypsin in the reaction system, causes a marked increase in both kcat and gm(agg) of tryptic hydrolysis of Z-Arg-Nan). Apparently, the phenomena is the main reason for the erroneous conclusions of Bresler et al. (1969, 1971), who stated : trypsin with an acylated active serine remains a highly efficient catalyst of amide hydrolysis. Finally, the data of Bresler et al. on tryptic hydrolysis and hydroxylaminolysis which contradict the acylenzyme mechanism of hydrolysis of the amide bond can be readily accounted for by chemistry of hydroxylamine, as was done by Bender et al. Epand andWilson (1963).Thus, the data obtained so far strongly support the acyl-enzyme mechanism of the hydrolysis of the amide bond catalyzed by trypsin and a-chymotrypsin.I n the late forties, a-chymotrypsin and trypsin were established to have, in addition to the amidase and peptidase activities, an esterase activity. Since then the question has arisen whether the enzymic hydrolysis of amide and esteric substrates proceeds via a similar mechanism. The problem was originally posed in a more general way: does the hydrolysis of esters and amides take place on one active site of the enzyme or are the esterase and amidase activities controlled each by a centre of its own. The first papers dealing with this problem appeared as early as 1949 [1,2]. I n these and subsequent papers Jansen and Balls with co-workers, as well as other authors (see [3]), showed that the phosphorylation of a-chymotrypsin or trypsin is accompanied by a simultaneous decrease in the esterase, amidase and peptidase activities. The fact of the existence of the common active site in a-chymotrypsin was also supported by the data on photooxidation of the enzyme [4] Abbreviations. Z-Arg-Nan, N-carboxbenzoxy-L-arginine m-nitroanilide ; Bz-Arg-Nan, N-benzoyl-DL-arginine p-nitroanilide; Bz-Arg-OEt, N-benzoyl-L-arginine ethyl ester.Enzyme. Trypsin (EC 3.4.4.4).35 Eur. J. Biochem., Vo1.38 in which a-chymotrypsin inactivates with respect to both esteric and amide substrates. However, these data only suggest that a-chymotryptic and tryptic hydrolyses of amide and esteric substrates occur on the ...

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supporting

confidence: 56%

“…I n these and subsequent papers Jansen and Balls with co-workers, as well as other authors (see [3]), showed that the phosphorylation of a-chymotrypsin or trypsin is accompanied by a simultaneous decrease in the esterase, amidase and peptidase activities. The fact of the existence of the common active site in a-chymotrypsin was also supported by the data on photooxidation of the enzyme [4] Abbreviations. Z-Arg-Nan, N-carboxbenzoxy-L-arginine m-nitroanilide ; Bz-Arg-Nan, N-benzoyl-DL-arginine p-nitroanilide; Bz-Arg-OEt, N-benzoyl-L-arginine ethyl ester.…”

supporting

confidence: 54%

The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

Березин¹,

Kazanskaya²,

Klyosov³

et al. 1973

European Journal of Biochemistry

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“…[37][38][39][40][41][42][43][44][45] Previously, photooxidation was used to study protein function through amino acid alterations. [46][47][48][49][50][51][52][53][54][55][56] The discovery that photooxidation could stabilize a protein solution 56 led to the development of this technology for biomedical purposes. The process was found to yield a biomaterial possessing many favorable properties for long-term implantable medical devices.…”

Section: Introductionmentioning

confidence: 99%

Calcification resistance, biostability, and low immunogenic potential of porcine heart valves modified by dye-mediated photooxidation

Moore

Adams

2001

J. Biomed. Mater. Res.

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The calcification potential, biostability, and immunogenic response of materials intended for long-term in vivo use, such as in heart-valve bioprostheses, are essential components of device performance. Here we explore these properties in photooxidized porcine heart valves. To study immunological sensitization, we injected tissue extracts intradermally into guinea pigs. Test and control animals received a challenge patch of the appropriate extract and were scored for dermal reactions. Neither cottonseed oil nor sodium chloride extracts of photooxidized heart-valve tissues caused any dermal inflammatory response. After implantation in the rat subcutaneous model for 90 days, the calcium content of 48-h-treated photooxidized cusp tissue [0.04 +/- 0.00 mg/g wet weight (gww)] was comparable to that of unimplanted control tissues (usually <1 mg/gww) and much lower than that of glutaraldehyde-treated controls (71 +/- 15 mg/gww). The porcine aortic wall calcium content (49 +/- 31 mg/gww) was comparable to that of glutaraldehyde-treated controls (59 +/- 8 mg/gww). Histologically, a time-dependent decrease in inflammation and vascularization with increasing photooxidation time was noted in the rat model along with an increase in the stability and organization of collagen bundles. In summary, porcine valve tissues treated by dye-mediated photooxidation were resistant to calcification, were biostable, and demonstrated a low immunogenic response, indicating potential for use in heart-valve bioprostheses.

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“…Photodynamic oxidation of histidine and other amino acids has been shown to occur (9-11). Weil et al (79)(80)(81), have been able to correlate the less of enzymatic activity with the photeoxidation of these amino acids in such enzymes as lysozyme, chymotrypsin and ribenuclease, presumably because the photooxidation alters the active site. This has been done more recently with lactic dehydrogenase (82).…”

Section: The Biochemistry Of Trypsinmentioning

confidence: 99%

Quantum Yield Studies of the Photodynamic Inactivation of Trypsin

Glad¹,

Spikes²

1964

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Photooxidation of crystalline chymotrypsin in the presence of methylene blue

Cited by 182 publications

References 25 publications

The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

Calcification resistance, biostability, and low immunogenic potential of porcine heart valves modified by dye-mediated photooxidation

Quantum Yield Studies of the Photodynamic Inactivation of Trypsin

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