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B515146e.dvi

www.rsc.org/pps | Photochemical & Photobiological Sciences A simple procedure for the photoregulation of chymotrypsin activity
Stephen Thompson,* Marie-Claude Fawcett, Lesley B. Pulman and Colin H. Self*
Received 25th October 2005, Accepted 9th January 2006
First published as an Advance Article on the web 19th January 2006
DOI: 10.1039/b515146e
A convenient and rapid method for the photo-regulation of the proteolytic enzyme a-chymotrypsin isdescribed. When a-chymotrypsin is coated with photolytic 1-(2-nitrophenyl)ethanol residues this notonly markedly reduces the capability of the enzyme to digest both of the small substratesN-benzoyl-L-tyrosine ethyl ester and N-succinyl-L-phenylalanine p-nitroanilide, but also completelyinhibits the enzyme’s proteolytic activity. The inactivated a-chymotrypsin can then be reactivated underphysiological conditions, when and where it is required, by exposure to UV-A light. These resultsfurther demonstrate that 1-(2-nitrophenyl)ethanol coated proteins can often be used as light sensitivebiological switches as a simple alternative to site directed procedures.
Introduction
of antibody activity15,16 further demonstrates that this NPE coating
procedure is generally applicable to protein photo-regulation.
Many important cellular and biological studies have depended on Such conjugates could be used as light controlled biological the synthesis and use of ‘caged’ compounds. On irradiation with switches in numerous fundamental and therapeutic applications.
light the photolabile protecting groups are removed and biological
activity is regenerated both when and where it is required.1,2
Unfortunately, however, relatively few studies have tackled
Experimental
the more complex goal of ‘caging’ proteins.3,4 It is extremely
difficult to photo-regulate the activity of enzymes unless the
a-Chymotrypsin (Type II) was obtained from Sigma, Poole, UK, enzyme is blocked in a site specific manner, and this often the substrates SPNA and BTEE from ICN Biochemicals, Thame, requires more complex techniques such as unnatural amino acid UK, and all other chemicals from Fluka, Poole, UK.
mutagenesis.5–8 Indeed it is often much easier to block an enzyme
modulator/catalyst than the enzyme itself.9,10 A non site-specific
Coupling of 1(2-nitrophenyl)ethanol groups
‘caging’ procedure which could be used to photo-regulate theactivity of enzymes would be a very useful biological tool.
1(2-Nitrophenyl)ethanol was coupled covalently to a-chymo- The serine proteases, thrombin, factor Xa and chymotrypsin trypsin (Fig. 1) as previously described for BSA and IgG.14–16
have been caged by the addition of cinnamate derivatives. These Ten ll of 1-(2-nitrophenyl)ethanol (NPE) was reacted at room were tailored to fit the enzyme’s active site and were subsequently temperature with 7.8 ll of di-phosgene in 250 ll of dry dioxan found to inhibit the enzyme by acylating the site’s essential serine in the presence of 5.2 ll pyridine as a catalyst. The mixture was residue.11–13 Some of the inactive acylated enzymes were not very
left for 15 min before unreacted materials were evaporated away stable and hydrolysed back to the active enzyme fairly rapidly.13
in a stream of nitrogen. The highly reactive 1-(2-nitrophenyl)- However others could be considered to be stable indefinitely. On ethoxycarbonylchloride was then resuspended in 250 ll dioxan UV-irradiation at 366 nm for 5–10 min up to 80% of the enzyme’s and 0, 5, 10, 20 and 50 ll aliquots of this solution were added to 1 ml portions of a 1.4 mg ml−1 solution of a-chymotrypsin We here discuss an alternative and simpler procedure for in 0.1 M NaHCO pH 8.3. The mixtures were left overnight for the reversible inhibition of a-chymotrypsin. This is based on a coupling to go to completion then dialysed against 0.9% NaCl at generically applicable method14 we have previously shown to be
4 C for 16 h. White insoluble reaction products were removed by
capable of reversibly inactivating antibodies.15,16 a-Chymotrypsin
micro-centrifugation for 15 min at 13 000 g and the NPE-coated is inactivated by simply coating it with photocleavable 1(2- chymotrypsin conjugates were left in the clear supernatant.
nitrophenyl)ethanol (NPE) residues via photolabile carbamate The protein concentration18 and the absorbance (OD280 nm)
linkages17 (Fig. 1). On irradiation with UV light at 365 nm
value of each sample were measured. As the absorbance of the 2-nitrobenzyl residues transform into unstable 2-nitroso uncoated a-chymotrypsin was known from the control sample, and derivatives3 which then break down into nitrosoacetophenone
a solution of 50 lg ml−1 NPE had an OD280 nm value of 1.0, the and CO releasing the enzyme in its natural form. This restoration amount of NPE, and hence the average number of NPE residues of enzyme activity, when taken in conjunction with the regulation bound to each a-chymotrypsin molecule could be estimated fromthe relative increase in the absorbance value of each sample. Itis assumed that the caging of the enzyme with NPE does not Diagnostic and Therapeutic Technologies, School of Clinical and Laboratory affect the protein assay. The fact that the OD values decrease Sciences, University of Newcastle upon Tyne, The Medical School, Fram- (through loss of NPE), whilst the protein concentration remains lington Place, Newcastle upon Tyne, UK NE2 4HH. E-mail: C.H.Self@ncl.ac.uk, Stephen.thompson@ncl.ac.uk; Fax: +44 0191 2226227; Tel: +44 constant, following irradiation and dialysis of the caged samples, 326 | Photochem. Photobiol. Sci., 2006, 5, 326–330
This journal is The Royal Society of Chemistry and Owner Societies 2006 The proposed reaction mechanism for the coupling and decoupling of 1(2-nitrophenyl)ethanol to proteins.
Enzymic activity
Photolysis of conjugates
Two small substrates were initially used in the determination of a- a-Chymotrypsin samples were diluted to 0.1 mg ml−1 in 0.9% chymotrypsin activity, N-benzoyl-L-tyrosine ethyl ester (BTEE)19
NaCl in quartz cuvettes. They were then irradiated with UV and N-succinyl-L-phenylalanine p-nitroanilide (SPNA).20 In both
light for a range of times at room temperature. After irradiation assays sample activity (both uncoated and NPE-coated samples), the samples were then dialysed for 16 h against 0.9% NaCl to was measured both before and after irradiation with UV light.
remove photocleaved products and the protein concentrations and The BTEE assay was carried out in a quartz cuvette. 500 ll of OD280 nm values were remeasured. The reduction in OD values 1.07 mM BTEE in 50% methanol was added to 300 ll of 80 mM to those of uncoated chymotrypsin not only demonstrated that Tris-HCl buffer pH 7.8 containing 100 mM CaCl . 200 ll of a the NPE was being removed, but also proved that its photolysed solution of each a-chymotrypsin sample (0.1 mg ml−1 in 0.9% reaction products were not binding to the chymotrypsin. A NaCl) was then added to the cuvette and the rate of reaction Spectroline EN-16/F UV lamp (Spectronics Corporation, New was measured at 256 nm for 90 s. The SPNA assay was carried York) with an emission peak of 365 nm was used as the source out in a 96 well ELISA plate. SPNA was first dissolved in 50% of the UV light. The total UV-A irradiance of the lamp was 5.45 methanol then an equal volume of 200 mM triethanolamine buffer mW cm−2 at the working distance of 0.3 cm.22 The a-chymotrypsin
(TRA) pH 7.8 containing 100 mM CaCl was added. 200 ll of this samples that were used to digest BSA were irradiated with a VL- solution was then pipetted into each well of an ELISA plate. 33 ll 206BL UV-A lamp (2 × 6 W tubes) which had a total UV-A of a 0.1 mg ml−1 solution of each a-chymotrypsin sample was irradiance of approximately 16 mW cm−2 at a distance of 1 cm.
then added to each well and the absorbance of the samples wasmeasured at 405 nm at 0, 15 and 30 min after their addition.
An average of 0, 1, 4, 7 and 9 NPE residues bound to eacha-chymotrypsin molecule on the addition of 0, 5, 10, 20 and50 ll NPE-carbonyl chloride to each a-chymotrypsin sample.
Proteolytic activity
The enzymic activity of each sample was then measured in three Bovine serum albumin (BSA) was added to both NPE-coated separate assays with each substrate and the results of a typical and uncoated a-chymotrypsin samples to determine how the NPE assay with each substrate are given in Tables 1 and 2. In all cases coating affected the ability of the enzyme to digest large protein enzymic activity is expressed as a percentage of that obtained with uncoated unirradiated native enzyme.
250 ll of uncoated and NPE-coated a-chymotrypsin samples (at When BTEE was used as the substrate (Table 1) the activity of 0.5 mg ml−1) were irradiated with UV-A light and 50 ll aliquots the enzyme was found to decrease from 100% to as little as 7% of were removed after 0, 2, 5 or 10 min. Each aliquot had 100 ll of the native value as the number of coupled NPE groups increased.
BSA (0.5 mg ml−1 in Tris buffer pH 7.8 as above) added to it and However when the NPE groups were removed by exposure to UV- the samples were then left for 15 min for digestion to proceed.
light the majority of the enzyme’s activity could be restored. The 100 ll of denaturing electrophoresis sample buffer21 containing
activity of the a-chymotrypsin sample coated with 1 NPE residue 10% SDS was then added and the samples were immediately boiled increased from 65 to 96% on 15 min UV-irradiation, whilst the for 5 min to stop the reaction. Equal quantities of each sample sample coated with 9 NPE residues increased from 7 to 78% (a 10 (30 ll) were then separated by discontinuous electrophoresis in 9% polyacrylamide gels.21 After staining with Coomassie blue dye
When SPNA was used as the substrate (Table 2) similar results the amount of undigested BSA remaining in each sample was were obtained. The enzyme activity progressively decreased as determined by scanning laser densitometry.
the number of NPE groups coating the enzyme increased and This journal is The Royal Society of Chemistry and Owner Societies 2006 Photochem. Photobiol. Sci., 2006, 5, 326–330 | 327
The photoregulation of a-chymotrypsin activity (using BTEE)a
relevant assay, was then examined. A new UV-A lamp was alsoused in an attempt to reduce irradiation times.
a-Chymotrypsin was again coated with 20 or 50 ll NPE. Table 3 shows that after 0, 2, 5 and 10 min irradiation an average of 5.4,1.1, 0.3 and 0 residues of NPE and 10.5, 2.9, 1.5 and 0.7 residues of NPE remained on each a-chymotrypsin molecule in each sample.
The vast majority of the NPE was therefore released by 2 min irradiation and very few residues were present after 5 min UV-A treatment. The amount of BSA digested by each sample is shown a The figures for a-chymotrypsin activity from each NPE-coated sample
in Fig. 2 and quantified in Table 4. The a-chymotrypsin sample represent the increase in OD 256 nm value (due to the release of N- coated with 5.4 residues was initially inhibited by around 50% but benzoyl-L-tyrosine) given by each sample expressed as a percentage of was restored to full activity by 2 min irradiation. The highly coated the increased OD 256 nm value given by control uncoupled unirradiated sample appeared to show total inhibition followed by the rapid and total recovery of a-chymotrypsin activity. Due to the importance ofthe latter result, another sample of highly coated a-chymotrypsin The photoregulation of a-chymotrypsin activity (using SPNA)a
was prepared (50 ll NPE, 11 residues of NPE per a-chymotrypsinmolecule). This was irradiated along with another aliquot of the previous highly coated sample. The results are shown in Fig. 3 andTable 5. Complete inhibition followed by rapid and total recovery of a-chymotrypsin activity was found in each sample.
a The figures for a-chymotrypsin activity from each NPE-coated sample
represent the increase in OD 405 nm value (due to p-nitroaniline release)
given by each sample expressed as a percentage of the increased OD 405 nm
value given by control uncoupled unirradiated a-chymotrypsin.
A comparison of the proteolytic activity of a-chymotrypsin and The removal of NPE residues from a-chymotrypsin by UV-A NPE coated a-chymotrypsin samples before and after irradiation withUV-A light. All lanes were loaded with 30 ll of sample containing identical amounts of BSA (6 lg). Lanes 1 and 12 were control lanes containingundigested BSA alone. Lanes 6 and 7 show the amount of BSA digested by uncoated chymotrypsin after 0 and 10 min irradiation. Lanes 2–5 showthe amount of BSA digested by NPE(5.4 residues)-chymotrypsin after 0, 2, 5 and 10 min irradiation. Lanes 8–11 show the amount of BSA digested by NPE(10.5 residues)-chymotrypsin after 0, 2, 5 and 10 min irradiation.
considerable enzymic activity could be restored on irradiation with Discussion
UV light for 15 min. This could be increased still further (especiallyin the samples coated with 7 or 9 residues) on 30 min irradiation.
The above data demonstrate that chymotrypsin can be coated As the above initial studies had shown that the NPE-coating with up to 11 residues of NPE which completely deactivates could reversibly inhibit the ability of a-chymotrypsin to digest the enzyme. This NPE coat can be rapidly removed, and the both small substrates, the ability of NPE-coated a-chymotrypsin enzyme reactivated, by illumination with light from a hand to digest a larger protein substrate (BSA), a more biologically held UV-A lamp. Small differences in coating are sometimes The photoregulation of a-chymotrypsin proteolytic activitya
a The figures for a-chymotrypsin activity from each NPE-coated sample represent the amount of BSA digested by each sample expressed as a percentage
of the amount of BSA digested by control uncoupled unirradiated a-chymotrypsin. Values are given for densitometry of the same samples on 2 different
gels.
328 | Photochem. Photobiol. Sci., 2006, 5, 326–330
This journal is The Royal Society of Chemistry and Owner Societies 2006 The photoregulation of two highly coated NPE-chymotrypsin The precise nature of this effect is to some extent unimportant, samplesa
as long as activity is regained on removal of the caging groups.
This work demonstrates that a-chymotrypsin as well as antibody preparations15,16,25 can be rendered inactive with a 2-nitrobenzyl
coat then reactivated at will by irradiation with UV light. Thissimple coating procedure operates efficiently in aqueous solutions, does not require site specific targeting or the synthesis of enzyme specific reagents,5–8,11–13 and photo-recovery of activity is very rapid
in therapeutic terms. Perhaps more importantly, the low power
a The figures for a-chymotrypsin activity from each NPE-coated sample
represent the amount of BSA digested by each sample expressed as
of the lamps means that reactivations can be carried out in the a percentage of the amount of BSA digested by control uncoupled presence of cells and tissues without damaging them. We did not examine how short photolysis times could be with a more powerful
lamp. Kossel et al.25 reduced their reactivation times from >3 h
with a hand held lamp, to seconds, utilising pulses from a focused
100 W mercury arc lamp. This allowed them to study synaptic
potentiation in brain slices.
The relatively simple nature of this procedure makes it an attractive starting point prior to attempting more complex sitespecific molecular biological approaches. We are convinced thatphoto-switching procedures such as this will play an essential partin both diagnostic and clinical medicine as well as providing auseful tool for a better understanding of basic cellular reactions.
We are currently using this photo-switching procedure to develop The digestion of BSA by two heavily coated NPE-chymotrypsin samples before and after irradiation with UV-A light. All lanes were again methods to deliver therapeutic proteins more accurately.
loaded with 30 ll of sample containing identical amounts of BSA (6 lg).
Lanes 1, 5 and 14 were control lanes containing undigested BSA alone.
Acknowledgements
Lanes 2–4 show the amount of BSA digested by uncoated chymotrypsinafter 0, 5 and 10 min irradiation. Lanes 6–9 and lanes 10–13 show the We thank British Petroleum for a grant through the BP Venture amount of BSA digested by two heavily coated NPE-chymotrypsin samples Research Unit and BioEnhancements Ltd for financial support.
(10.5 and 11 residues) after 0, 2, 5 and 10 min irradiation respectively.
obtained, e.g. 5.4/7 residues by the addition of 20 ll of the 1- References
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