Screening of Inhibitors for Mushroom Tyrosinase Using Surface

Subscriber access provided by BRIGHAM YOUNG UNIV

Article

Screening of inhibitors for mushroom tyrosinase using Surface Plasmon Resonance Sushama Patil, Srinivas Sistla, and Jyoti Prafulla Jadhav J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5039585 • Publication Date (Web): 05 Nov 2014 Downloaded from http://pubs.acs.org on November 17, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Screening of inhibitors for mushroom tyrosinase using Surface Plasmon Resonance

2 3 4 5 6

Sushama Patil†, Srinivas Sistla‡, Jyoti Jadhav*† †

7

Running title: Binding affinity studies of tyrosinase inhibitors

‡

Department of Biotechnology, Shivaji University, Kolhapur- 416004, GE Healthcare Life Sciences, John F Welch Technology Centre, EPIP, Phase 2, Whitefield Road, Bangalore, India-560048

8 9

*Corresponding author

10

Prof. Mrs. Jyoti P. Jadhav

11

Professor and Head

12

Department of Biotechnology,

13

Shivaji University, Vidyanagar,

14

Kolhapur 416004, India

15

E-mail: [email protected]

16

Tel.: +91 231 2609365

17

Fax: +91 231 1691533

18 19

20

21

22

23

24 25 26 1

ACS Paragon Plus Environment


27

Page 2 of 29

Abstract

28

Tyrosinase inhibitors have been used as whitening or antihyperpigment agents because of their

29

ability to suppress dermal-melanin production. In this present study, screening and kinetic evaluation

30

of various small molecules was studied on mushroom tyrosinase (MT) using surface plasmon

31

resonance. The binding constant KD (M) values obtained for tannic acid, phloroglucinol, saffron,

32

catechol and pyrogallol are 1.213 × 10-4, 7.136 × 10-5, 3.111 × 10-5, 1.557 × 10-5 and 7.981 × 10-6 M

33

respectively. Pyrogallol has been found to display high affinity for MT whereas catechol, saffron and

34

phloroglucinol have been found to bind with low affinity. MT shows considerable changes in the

35

secondary structure in presence of inhibitors. The study reveals the Biacore/SPR sensor’s ability for

36

rapid identification and characterization of inhibitors for MT. Methodology described here can be used

37

to rapidly screen and optimize various leads compounds for other enzymes and elucidate structure

38

function inter-relationships between various enzymes.

39

Keywords: Mushroom tyrosinase, inhibitors, surface plasmon resonance

40 41

2


Page 3 of 29

42 43


Introduction

44

Tyrosinase (EC1.14.18.1), a multifunctional copper-containing oxygenase, is widely distributed

45

in nature. It catalyzes the hydroxylation of a monophenol and the conversion of o-diphenols to the

46

corresponding o-quinones1. These quinones are highly reactive compounds that can polymerize

47

spontaneously to form melanin. Products formed as a result of tyrosinase activity are precursors for

48

skin pigmentation, defense and protective mechanisms in plants and fungi, and leads to pigmentation in

49

flowers2-6. These precursors from tyrosinase activity may also lead to deleterious effects like browning

50

reactions in fruits and vegetables and black spotting of shrimp and lobsters7, 8. Various inhibitors of

51

mushroom tyrosinase are reported such as, aromatic aldehydes9, 10 acids11, tropolone12 and kojic acid13.

52

Changes in tyrosinase activity can be measured using conventional spectrophotometric

53

methods by detection of the substrate turnover at different wavelengths14-16. The most accepted dye

54

based or tracer based assays are Besthorn's hydrazone17, 18 cysteine19 or proline20. Many electrochemical

55

methods are based on measurement of oxygen consumption21 or product formation is reported in the

56

literature22-26. However these assays will produce much false positive identification of compounds as

57

they are color based or dye based reactions. Identification and characterization of lead compounds by

58

SPR is a very rapid and promising alternative.

59

Surface plasmon resonance (SPR) with coupling chemistries for enzyme immobilization on the

60

sensor surface facilitates detection and screening of various inhibitors27. SPR is an optical technique

61

that analyses changes in refractive index based on alterations occurring in the dielectric medium within

62

500 nm from a metal gold surface28. SPR makes possible real-time, label-free detection of biomolecular

63

binding events such as ligand–receptor coupling, antibody–antigen interactions, and protein–DNA

64

interactions. Detection of tyrosinase inhibitors using SPR has been reported recently29, 30. In this present

65

study, screening of small molecules as inhibitors of mushroom tyrosinase was studied by using SPR. 3



66

Material and methods

67

Chemicals and reagents

68

Sensor

chips

CM5,

N-ethyl-N’-(dimethylaminopropyl)-carbodiimide

Page 4 of 29

(EDC),

N-

69

hydroxysuccinimide (NHS), ethanolamine HCl, sampling vials, were obtained from GE Healthcare Life

70

Sciences, Uppsala, Sweden. Mushroom tyrosinase was purchased from Sigma chemical company (St.

71

Louis, MO, USA). All other chemicals were from Himedia India Pvt ltd, India with highest purity and

72

analytical grade. Milli Q (Milipore, USA) water was used for preparing buffers and reagents.

73

Enzyme activity assay

74

Monophenolase and diphenolase activities of mushroom tyrosinase was performed using L-

75

Tyrosine and L-DOPA respectively by measuring the dopachrome accumulation at 475 nm (ε

76

dopachrome = 3400 M−1 cm−1)31, 32 before immobilization on chip surface.

77

Surface plasmon resonance (SPR) studies

78

SPR interaction analyses were performed using a Biacore X100 optical biosensor (GE

79

Healthcare Life Sciences, Bangalore, India). SPR measurements were carried out in phosphate buffer

80

saline [0.1 M phosphate buffer with 27 mM KCl and 1.37 M NaCl 0.005 % polysorbate 20 pH 7.4], and

81

stock solutions were diluted in the same buffer. Data was collected with the Biacore control software

82

version 2.0. Experiments were performed by monitoring the refractive index changes as a function of

83

time under constant flow rate of 30 µl/min. The relative amount of inhibitor bound to the tyrosinase

84

was determined by measuring the net increase in refractive index over time compared to control

85

running buffer. There is an inline subtraction of reference surface during the run. This change is usually

86

reported in response units (RU).

87

Enzyme immobilization

4


Page 5 of 29


88

Mushroom tyrosinase dissolved (50 µg ml-1) in 0.1 M sodium acetate buffer pH 4.5 was

89

immobilized to a CM5 chip using amine coupling. Using a flow rate of 10 µl min-1, the surface of flow

90

cell was activated for 7 min using a 1:1 mixture of 100 mM N-ethyl-N’-(dimethylaminopropyl)-

91

carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS) (both dissolved in water), and

92

subsequently tyrosinase was injected for 7 min, and residual activated carboxy methyl groups on the

93

surface were blocked by a 7 min injection of 1M ethanolamine, pH 8.5. A total of 2353 (RU) of

94

tyrosinase was immobilized. The activity of enzyme after immobilization was confirmed by plotting RI

95

(RU) response against concentration of substrate (L-DOPA and L-tyrosine).

96

Screening of different small molecules

97

For this study, one of the flow cell was blank immobilized (without protein) for using as a

98

reference. Different analytes like, monohydroxy, dihydroxy and trihydroxy phenols were screened

99

against mushroom tyrosinase. Flow rate was maintained constant throughout the binding (10 µl/min)

100

and kinetics experiment (30 µl/min), contact time and dissociation time was kept at 120 s. Regeneration

101

was carried out with 10 mM glycine HCl pH 2.5 for 30 s and at 30 µl/min. Kinetics was performed with

102

various concentrations of compounds (12.5 µM to 200 µM) in a single cycle kinetics33, 34 or multi-cycle

103

kinetics mode. The data analysis was done with Biacore X100 evaluation software ver 2.0.1 and data

104

was fit to 1:1 binding model or two state fit.

105

Circular dichroism (CD) spectroscopy

106

The CD spectra were recorded on a Jasco J-815 CD Spectropolarimeter to give the content of

107

regularly secondary structure in mushroom tyrosinase. Protein solutions 0.05 mg ml-1 was prepared in

108

the 0.1 mM PBS buffer at pH 6.8. Scans were performed at 20 °C using 0.1 cm path length quartz

109

cuvette with 8 sec differential integration time at a scan rate of 50 nm min-1. The protein solutions of

110

0.05 mg ml-1 were incubated with and without pyrogallol, phloroglucinol, tannic acid, saffron and 5



Page 6 of 29

111

catechol at concentration of 200 µM for 15 min and were used to obtain the spectra. All spectra were

112

collected in a triplicate from 200 to 280 nm and a background corrected against buffer blank. The

113

results were expressed as molar ellipticity (◦cm2 dmol-1) based on a mean amino acid residue weight

114

(MRW) of mushroom tyrosinase. The molar ellipticity was determined as [θ obs] = (100 × (MRW) ×

115

θobs/cl), where θ is the observed ellipticity in degrees at a given wavelength, c is the protein

116

concentration in mg/ml and l is the length of the light path in cm35.

117

Result and discussion

118

It has been recently discovered that various dermatological disorders such as age spots and

119

freckles are caused due to hyperpigmentation1. Hyperpigmentation in human skin and enzymatic

120

browning in fruits are not desirable36. Thus, hyperpigmentation becomes a major problem in the food

121

industry and one of the main causes of quality loss during post harvest handling and processing31. The

122

regulation of melanin synthesis via the inhibition of tyrosinase is a current area of research in the

123

context of preventing hyperpigmentation37. As a result, tyrosinase inhibitors have become increasingly

124

important in food, medical and cosmetic industries38, 39.

125

Various tyrosinase-based biosensors have been developed in the past few years22-26. Using

126

these sensors, the detection of the large group of phenolic compounds as tyrosinase inhibitors was

127

possible. However, low sensitivity is one of their major drawbacks40. In the present paper, the affinity

128

and binding kinetics of various small molecules towards mushroom tyrosinase using surface plasmon

129

resonance has been evaluated. In addition, this sensor has ability to detect the number of different

130

inhibitors of enzyme in a single run with high sensitivity and accuracy. Label free interactions are more

131

sensitive and less error prone compared with the conventional spectroscopic methods which are based

132

on the color formation.

6


Page 7 of 29


133

The screening results showed that the affinity towards mushroom tyrosinase varies from

134

molecule to molecule which is clearly observed from Figure 1. Many compounds showed high affinity

135

for tyrosinase but possess low stability. Low stability compounds were found to display a higher

136

dissociation rate. Based on binding studies various small molecules like catechol, pyrogallol, saffron,

137

tannic acid and phloroglucinol were selected further for affinity and kinetic analysis against mushroom

138

tyrosinase. This study shows that pyrogallol has a higher binding affinity for mushroom tyrosinase with

139

KD values of 7.981 ×10-6 M. The data with different concentrations was fit to two state equation and

140

shown in the Table 1. Pyrogallol has higher rates of association and dissociation and a higher

141

equilibrium dissociation constant as evident from Figure 2. Pyrogallol is trihydroxy phenol: it perhaps

142

binds more rapidly as compared to L-DOPA and L-tyrosine which are dihydroxy and monohydroxy

143

phenols, respectively. Order of tyrosinase action on phenolic substrates follows the order monohydroxy

144

˃ dihydroxy ˃ trihydroxy phenols. The KD value for L-DOPA was reported by us before32. L-DOPA

145

was also run between inhibitors during the kinetic runs to ascertain the activity of tyrosinase. The

146

activity of enzyme after immobilization was proportional with increasing concentration of L-DOPA

147

and L-tyrosine (Figure 3) which confirmed the stability and reactivity of mushroom tyrosinase after

148

immobilization. Pyrogallol was reported as suicide inhibitor of tyrosinase and predicted to bind with

149

high affinity41. The low affinity of catechol (1.557 ×10-5 M) (Figure 4) compared to pyrogallol may be

150

due to catechol follows ‘cresolase presentation’. Catechol being oxidized to form a product able to

151

undergo deprotonation and reductive elimination, resulting in inactivation of the enzyme through the

152

formation of copper at the active site. However, since the OH groups are equivalent, the probability of

153

catalysis and inactivation increases in case of pyrogallol (trihydroxy phenol) and catechol (o-

154

diphenol)42. Catechol showed fast association and slow dissociation time as it is a substrate for

155

tyrosinase (Table 1). 7



Page 8 of 29

156

Saffron, phloroglucinol and tannic acid have a low binding affinity towards mushroom

157

tyrosinase with KD values of 3.111 ×10-5, 7.136 ×10-5 and 1.213 ×10-4 M, respectively (Table 1). Also

158

these three compounds showed slow association and fast dissociation in comparison with pyrogallol

159

and catechol (Table 1). The binding kinetics for saffron (Figure 5), phloroglucinol (Figure 6) and tannic

160

acid (Figure 7) with 1:1 fit clearly indicate almost similar association and dissociation rate constants

161

which perhaps suggests a similar mode of inhibition. The key component in saffron is crocin, is

162

reported as a potent inhibitors of the tyrosinase32. Also, phloroglucinol (IC50 ˃ 300 µM) and tannic acid

163

are reported as inhibitors of mushroom tyrosinase43, 44. Inhibition of tannic acid is a reversible reaction.

164

In order to understand the, structural changes of the mushroom tyrosinase in the presence of

165

inhibitors was studied by CD spectroscopy technique. As illustrated in Figure 8, results in signiﬁcant

166

differences in the secondary structure of the native enzyme and mushroom tyrosinase in presence of

167

inhibitors. CD studies also confirm secondary structural changes analyzed using SPR. The % changes

168

in alpha helix in presence of inhibitors have been given (Table 2).

169

A number of tyrosinase inhibitors have earlier been reported to decrease hyperpigmentation

170

resulting from the enzyme action. Most of these tyrosinase inhibitors were mono, di and trihydroxyl

171

phenols, flavonoids, and peptides36, 45. These reported activators and inhibitors are phenols and other

172

common cosmetic ingredients well studied on mushroom tyrosinase.

173

Biacore sensor has the ability to detect changes in protein conformation in addition to binding

174

of inhibitors. Direct detection of low molecular-weight analytes by SPR has been reported for binding

175

of Ca2+ ions to transglutaminase46 and sulfonamide inhibitors to carbonic anhydrase47. The present

176

study demonstrates for the first time direct detection of tyrosinase inhibitors binding using an SPR

177

sensor. The study proves the ability of this sensor to screen the binding ability of different types of

178

inhibitors towards mushroom tyrosinase at various concentrations and also to be used for the screening 8


Page 9 of 29


179

of new tyrosinase inhibitors. In addition, the sensor was relatively simple to make and did not require

180

the use of antibodies to detect small size inhibitors employed in the study. Once the enzyme is

181

immobilized on the sensor surface it will be useful for a long time through proper storage. Moreover,

182

by immobilizing other enzymes or cell receptors of interest on the SPR sensor surface, their response

183

on exposure to inhibitors and ligands can also be assessed, resulting in the development of novel and

184

rapidly sensing biosensors. Other, undesirable properties relate more to how the target and ligand

185

interact. One such mechanism, recently described by McGovern et al.48, 49 involves the formation of

186

ligands into aggregates of 30-400 nm in diameter. These aggregates were proposed to inhibit either by

187

absorption onto the surface of enzymes or by incorporating enzymes within them. Inhibitors acting in

188

this manner were termed “promiscuous” inhibitors, as they appeared to inhibit a number of unrelated

189

enzymes, although it is likely that there are other mechanisms by which compounds may inhibit

190

numerous enzymes. McGovern et al. 48, 49 identified and characterized a number of these “promiscuous”

191

inhibitors by testing for properties that distinguished them from classical 1:1 reversible inhibitors.

192

In conclusion, pyrogallol, catechol, saffron and phloroglucinol and tannic acid had anti-

193

tyrosinase activity, but showed difference in their binding affinity for the tyrosinase. Therefore, this

194

enzyme-based biosensor has the potential to be used in the detection and screening of new inhibitor

195

drug candidates. Another advantage is that label free interactions are more sensitive compared with the

196

spectroscopic methods. Inhibitors may therefore have good potential as antibrowning agents to be

197

applied in food industries as well as in cosmetics. The sensors can be used for high-throughput

198

screening of potential pharmaceutical drug candidates.

199 200 201

Acknowledgements

202

acknowledged. Prof. Jyoti Jadhav wishes to thank Interdisciplinary Programme for Life Sciences

BSR meritorious fellowship to Ms. Sushama A. Patil from UGC, New Delhi, India is gratefully

9



Page 10 of 29

203

sponsored by Department of Biotechnology Government of India under DBT-IPLS programme. All

204

the authors would like to thank Maharashtra State Govt. for Golden Jubilee funding. The authors are

205

thankful to IIT- Mumbai for CD spectroscopy facility.

206 207

Conflict of interest: The authors have declared no conflict of interest.

208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 10


Page 11 of 29


226

References

227

(1) Yan, Q.; Cao, R.; Yi, W.; Chen, Z.; Wen, H.; Ma, L.; Song, H. Inhibitory effects of 5-benzylidene

228

barbiturate derivatives on mushroom tyrosinase and their antibacterial activities. Euro. J. Med.

229

Chem. 2009, 44, 4235–43.

230 231 232 233

(2) Ando, H.; Kondoh, H.; Ichihashi, M.; Hearing, V. J. Approaches to identify inhibitors of melanin biosynthesis via quality control of tyrosinase. J Invest. Dermatol. 2007, 127, 751-61. (3) Mayer, A. M. Polyphenol oxidases in plants and fungi: Going places? Phytochem. 2006, 67, 231831.

234

(4) Selinheimo, E.; NiEidhin, D.; Steffenson, C.; Nielson, J.; Lomascolo, A.; Halaouli, S.; Record, E.;

235

O’Beirne, D.; Buchert, J.; Kruus, K. Comparison of the characteristics of fungal and plant

236

tyrosinases. J Biotechnol. 2007, 130, 471-80.

237

(5) Gandia-Herrero, F.; Escribano, J.; Garcia-Carmona, F. Betaxanthins as substrates for tyrosinase: An

238

approach to the role of tyrosinase in the biosynthetic pathway of betalains. Plant Physiol. 2005,

239

138, 421-32.

240 241 242 243 244 245 246 247

(6) Yoruk, R.; Marshall, M. Physicochemical properties and function of plant polyphenol oxidase: A review. J Food Biochem. 2003, 27, 361-422. (7) Walker, J. R. L.; Ferrar, P. H. The control of enzymatic browning in foods. Chem. Indust. 1999, 16 836-39. (8) Kim, J.; Marshall, M. R.; Wei, C. Polyphenoloxidase. In Sea Food Enzymes, Haard, N.F.; Simpson, B.K.; Eds., Marcel Dekker: New York, NY, USA. 2000, 271-315. (9) Huang, X. H.; Chen, Q. X.; Wang, Q.; Song, K. K.; Wang, J.; Sha, L. Inhibition of the activity of mushroom tyrosinase by alkylbenzoic acids. Food Chem. 2006a, 94, 1–6.

11



Page 12 of 29

248

(10) Huang, X. H.; Chen, Q. X.; You, M. S.; Wang, Q.; Song, K. K.; Wang, J. Inhibitory effects of

249

fluorobenzaldehydes on the activity of mushroom tyrosinase. J of Enz. Inhibition and Med.

250

Chem. 2006b, 21, 413–18.

251

(11) Wang, Q.; Shi, Y.; Song, K. K.; Guo, H. Y.; Qiu, L.; Chen, Q. X. Inhibitory effects of 4–

252

halobenzoic acids on the diphenolase and monophenolase activity of mushroom tyrosinase. The

253

Prot. J. 2004, 23, 303–08.

254 255 256 257

(12) Espin, J. C.; Wichers, H. J. Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone. J of Agri. and Food Chem. 1999, 47, 2638–44. (13) Lee, Y. S.; Park, J. H.; Kim, M. H.; Seo, S.H.; Kim, H. J. Synthesis of tyrosinase inhibitory kojic acid derivative. Archiv der Pharmazie. 2006, 339, 111–14.

258

(14) Jacobson, G. M.; Iskandar, R.; Jacobsohn, M. K. Activity of mushroom tyrosinase on catechol and

259

on a catechol estrogen in an organic solvent. Biochim. Biophys. Acta. 1993, 1202, 317-24.

260

(15) Fling, M.; Horowitz, N. H.; Heinemann, S. F. The isolation and properties of crystalline tyrosinase

261 262 263 264 265 266 267 268 269

from Neurospora. J Biol. Chem. 1963, 238, 2045-53. (16) Waite, J. H. Calculating extinction coefficients for enzymatically produced o-quinones. Anal. Biochem. 1976, 75, 211-18. (17) Sussman, A. S. A comparison of the properties of two forms of tyrosinase from Neurospora crassa. Arch. Biochem. Biophys. 1961, 95, 407-415. (18) Pfifferi, P. G. Baldassari, L. A spectrophotometric method for the determination of the catecholase activity of tyrosinase by Besthorn's hydrazone. Anal. Biochem. 1973, 52, 325-35. (19) Gauillard, F.; Richard-Forget, F.; Nicolas, J. New spectrophotometric assay for polyphenol oxidase activity. Anal. Biochem. 1993, 215, 59-65.

12


Page 13 of 29

270 271 272 273 274 275


(20) Rezepecki, L. M.; Waite, J. H. A chromogenic assay for catecholoxidases based on the addition of L-proline to quinones. Anal. Biochem. 1989, 179, 375-81. (21) Mayer, A. M.; Harel, E.; Ben-Shaul, R. Assay of catechol oxidsase-a critical comparison of methods. Phytochem. 1966, 5, 783-89. (22) Hu, X.; Leng, Z. Determination of cyanide using a tyrosinase amperometric biosensor with catechol as substrate. Analyst. 1995, 120, 1555-57.

276

(23) Besombes, J. L.; Cosnier, S.; LabbeÂ, P.; Reverdy, G. A biosensor as warning device for the

277

detection of cyanide, chlorophenols, atrazine and carbamate pesticides. Anal. Chim. Acta. 1995,

278

311, 255-63.

279

(24) Macholan, L. Phenol-sensitive enzyme electrode with substrate cycling for quantification of

280

certain inhibitory aromatic acids and thio compounds. Collect. Czech. Chem. Comm. 1990, 55,

281

2152-60.

282

(25) Hasebe, Y.; Oshima, K.; Takise, O.; Uchiyama, S. Chemically amplified kojic acid responses of

283

tyrosinase-based biosensor, based on inhibitory effect to substrate recycling driven by

284

tyrosinase and L-ascorbic acid. Talanta. 1995, 42, 2079-85.

285

(26) Wang, J.; Nascimento, V. B.; Kane, S. A.; Rogers, K.; Smyth, M. R.; Angenes, L. Screen-printed

286

tyrosinase-containing electrodes for the biosensing of enzyme inhibitors. Talanta. 1996, 43,

287

1903-07.

288

(27) Milkani, E.; Christopher, R.; Lambert, W.; Grant, M. G. Direct detection of acetylcholinesterase

289

inhibitor binding with an enzyme-based surface plasmon resonance sensor. Anal. Biochem.

290

2011, 408, 212–19.

291 292

(28) Homola, J.; Yee, S. S.; Gauglitz, G. Surface plasmon resonance sensors: review. Sensors and Actuators B. 1999, 54, 3–15. 13



293 294 295 296

Page 14 of 29

(29) Germanas, J. P.; Wang, S.; Miner, A.; Hao, W.; Ready, J. M. Discovery of small-molecule inhibitors of tyrosinase. Bioorganic and Med. Chem. Lett. 2007, 17, 6871–75. (30) Lee, H. Y.; Park, S. B. Surface modification for small-molecule microarrays and its application to the discovery of a tyrosinase inhibitor. Mol. BioSyst. 2011, 7, 304–10.

297

(31) Shi, Y.; Chen, Q.; Qin, W.; Song, K.K.; Qiu, L. Inhibitory effects of cinnamic acid and its

298

derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase. Food

299

Chem. 2005, 92, 707–12.

300 301 302 303 304 305 306 307 308 309

(32) Patil, S.A.; Sistla, S.; Jadhav, J. P. Evaluation of crocin and curcumin affinity on mushroom tyrosinase using surface plasmon resonance. Biol. Macromol. 2014, 65, 163– 66. (33) Karlsson R.; Katsamba, P. S.; Nordin, H.; Pol, E.; Myszka, D. G. Analyzing a kinetic titration series using afﬁnity biosensors. Anal. Biochem. 2006, 349, 136–47. (34) William, P.; Carmelo, D. P. Simulated single-cycle kinetics improves the design of surface plasmon resonance assays. Talanta. 2013, 114, 211–16. (35) Yang, J.T.; Wu, C.S.; Martinez, H.M. Calculation of protein conformation from circular dichroism. Meth. Enzymol., 1986, 130, 208-69. (36) Kima, Y.; Uyamab, H. Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. Cellular Mol. Life Sci. 2005, 62, 1707–23.

310

(37) Li, Z.; Huachen, L.; Yu, X.; Hu, Y.; Song, K.; Wangzhou, X.; Chen, Q. Inhibition kinetics of

311

chlorobenzaldehyde thiosemicarbazones on mushroom tyrosinase. J of Agri. Food Chem. 2010,

312

58, 12537-40.

313

(38) Fenoll, L.; Rodríguez-López, J.; García-Sevilla, F.; García-Ruiz, P.; Varón, R.; García-Cánovas, F.

314

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols

14


Page 15 of 29


315

and diphenols generating highly unstable o-quinones. Biochim. Biophys. Acta. 2001, 1548, 1–

316

22.

317 318

(39) Han, P.; Chen, C.; Zhang, C.; Song, K.; Zhou, H.; Chen, Q. Inhibitory effects of 4-chlorosalicylic acid on mushroom tyrosinase and its antimicrobial activities. Food Chem. 2008, 107, 797-803.

319

(40) Streffera, K.; Kaatzb, H.; Bauera, C. G.; Makowera, A.; Schulmeisterc, T.; Schellera, F. W.;

320

Peterb, M. G., Wollenbergera, U. Application of a sensitive catechol detector for determination

321

of tyrosinase inhibitors. Anal. Chimica. Acta. 1998, 362, 81-90.

322

(41) Munoz-Munoz, J. L.; Garcia-Molina, F.; Garcia-Ruiz, P. A.; Molina-Alarcon, M.; Tudela, J.;

323

Garcia-Canovas, F.; Rodriguez-Lopez, J. N. Phenolic substrates and suicide inactivation of

324

tyrosinase: kinetics and mechanism. Biochem. J. 2008, 416, 413–40.

325

(42) Munoz-Munoz, J. L.; Garcia-Molina, F.; Varon, R.; Garcia-Ruız, P. A.; Tudela, J.; Garcia-

326

Canovas, F.; Rodrıguez-Lopez, J. N. Suicide inactivation of the diphenolase and

327

monophenolase activities of tyrosinase. IUBMB Life. 2010, 62, 539–47.

328

(43) Yoon, N. Y.; Eom, T. K.; Kim, M. M.; Kim, S. K. Inhibitory effect of phlorotannins isolated from

329

Ecklonia cava on mushroom tyrosinase activity and melanin formation in mouse B16 f 10

330

melanoma cells. J Agric Food Chem. 2009, 57, 4124-29.

331 332

(44) Xin, Q. L.; Hao, H.; Qingxi, C. The inhibitory effect on mushroom tyrosinase by tannic acid. J of Xiaman Uni. Nat. sci. 2005, 44, 839-842.

333

(45) Song, Y. H. Why tyrosinase for treatment of melanoma. Lancet. 1997, 350, 82-83.

334

(46) Gestwicki, J. E.; Hsieh, H. V.; Pitner, J. B. Using receptor conformational change to detect low

335 336 337

molecular weight analytes by surface plasmon resonance. Anal. Chem. 2001, 73, 5732–37. (47) Myszka, D. G. Analysis of small-molecule interactions using Biacore S51 technology. Anal. Biochem. 2004, 329, 316–23. 15



Page 16 of 29

338

(48) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A common mechanism underlying

339

promiscuous inhibitors from virtual and high-throughput screening. J. Med Chem. 2002, 45,

340

1712-22.

341 342

(49) McGovern, S. L.; Shoichet, B. K. Kinase inhibitors: Not just for kinases anymore. J. Med. Chem. 2003, 46, 1478-83.

343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 16


Page 17 of 29


361

Figure captions

362

Figure 1 Binding sensogram of various compounds towards immobilized mushroom tyrosinase (2353

363

RU), enzyme concentration was 50 µg ml-1 in 0.1 M sodium acetate buffer pH 4.5. Increasing

364

concentration for each compound (125 µM, 250 µM, and 500 µM) were injected simultaneously

365

over surface of immobilized enzyme and the total cycles ranges from 1 to 101 are as buffer (1-5),

366

followed by L-DOPA, catechol, L-tyrosine, phloroglucinol, ABTS, o-toludine, caffic acid,

367

gallic acid, guaiacol, syringaldehyde, pyrogallol, p-hydroxybenzoic acid, orcine, tannic acid,

368

resorcinol, saffron, veratrol, vertryl alcohol, o-dianisidine hydroquinone, curcumin, glutathione,

369

o-cresol, p-cresol, NaCl, sodium azide, ascorbic acid, kojic acid, thioglycolic acid, 2, 6

370

dimethoxybenzoic acid, m-cresol, crocin etc (respective 3 cycles for each analyte). Flow rate

371

was maintained constant throughout the binding (10 µl/min).

372

Figure 2 Binding sensogram for pyrogallol interaction with immobilized mushroom tyrosinase

373

Increasing concentration of pyrogallol from 6.25, 12.5, 25, 50 and 100 µM were injected over

374

the enzyme surface. Flow rate (30 µl/min) contact time and dissociation time was kept at 120 s.

375

Regeneration was carried out with 10 mM glycine HCl pH 2.5 for 30 s and at 30 µl/min. The

376

data analysis was done with Biacore X100 evaluation software ver 2.0.1 and data was fit to two

377

state.

378 379

Figure 3 The activity of enzyme after immobilization was confirmed by plotting RI (RU) response against concentration of substrate for L-DOPA (--♦--) and L-tyrosine (--■--).

380

Figure 4 Binding sensogram for catechol interaction with immobilized mushroom tyrosinase.

381

Increasing concentration of catechol from 12.5, 25, 50, 100 and 200 µM were injected over the

382

enzyme surface. Flow rate (30 µl/min) contact time and dissociation time was kept at 120 s.

383

Regeneration was carried out with 10 mM glycine HCl pH 2.5 for 30 s and at 30 µl/min. The 17



Page 18 of 29

384


385

state.

386

Figure 5 Binding sensogram for saffron interaction with immobilized mushroom tyrosinase. Increasing

387

concentration of Saffron from 12.5, 25, 50, 100 and 200 µM were injected over the enzyme

388

surface. Flow rate (30 µl/min) contact time and dissociation time was kept at 120 s.

389


390


391

state. The resulting equilibrium dissociation constants are reported in Table 1.

392

Figure 6 Binding sensogram for phloroglucinol interaction with immobilized mushroom tyrosinase.

393

Increasing concentration of Phloroglucinol from 12.5, 25, 50, 100 and 200 µM were injected

394

over the enzyme surface. Flow rate (30 µl/min) contact time and dissociation time was kept at

395

120 s. Regeneration was carried out with 10 mM glycine HCl pH 2.5 for 30 s and at 30 µl/min.

396

The data analysis was done with Biacore X100 evaluation software ver 2.0.1 and data was fit to

397

two state. The resulting equilibrium dissociation constants are reported in Table 1.

398

Figure 7 Binding sensogram for tannic acid interaction with immobilized mushroom tyrosinase.

399

Increasing concentration of Tannic acid from 6.25, 12.5, 25, 50 and 100 µM were injected over

400

the enzyme surface. Flow rate (30 µl/min) contact time and dissociation time was kept at 120 s.

401


402


403

state. The resulting equilibrium dissociation constants are reported in Table 1.

404 405

Figure 8 CD spectrum of mushroom tyrosinase without ( (

), tannic acid (

), saffron (

) and catechol (

) and with pyrogallol (

), phloroglucinol

) at 200 µm concentration each.

406 18


Page 19 of 29

407


Figures

408

409 410 411

Figure 1

412

19



Page 20 of 29

413 RU 180

R e s pons e

130

80

30

-20 -500

414

0

500

1000 Tim e

1500

2000

2500 s

415 416

Figure 2

417 418 419 420 421 422 423

20


Page 21 of 29


424 3.5

L-DOPA

L-tyrosine

3

RI (RU)

2.5 2 1.5 1 0.5 0 0

425

200

400

600

800

Concentration (µM)

426 427

Figure 3

428

21



Page 22 of 29

429 430 431 432 RU 25 20 15 10 R e s pons e

5 0 -5 -10 -15 -20 -25 -500

433

0

500

1000 Tim e

1500

2000

2500 s

434 435

Figure 4

436 437 438 439 440 441 442 443 444 445

22


Page 23 of 29


RU 20 15 10

R e s p on s e

5 0 -5 -10 -15 -20 -25 -500

446

0

500

1000 Tim e

1500

2000

2500 s

447 448 449

Figure 5

450 451 452 453 454 455 456 457 458 459 460 461 462

23



Page 24 of 29

RU 15

10

5

Response

0

-5

-10

-15

-20 -500

463

0

500

1000

1500

Tim e

2000

2500 s

464 465

Figure 6

466 467 468

24


Page 25 of 29


469 470 RU 5000

4000

3000

Response

2000

1000

0

-1000

-2000 -500

471

0

500

1000

1500

2000

2500 s

Tim e

472 473

Figure 7

474

25



Page 26 of 29

475

476 477 478

Figure 8

479

26


Page 27 of 29


480

Tables

481 482

Table 1 Interaction kinetics of mushroom tyrosinase with different inhibitors fit to two state model using Biacore evaluation software 483 Inhibitor

ka1(1/Ms)

kd1(1/s)

Ka2(1/s)

Kd2(1/s)

KD (M) 484

Tannic acid

Phloroglucinol

397.4±21

0.08624

8.708×10-4

0.001103

485 1.213×10-4±0.8

±0.0033

±4.1×10-5

±6.7×10-5

486

0.003219

7.136×10-5±2 487

±7.5×10-5

488

0.3582±0.019 5.208×10-4

4321±41

±5.2×10-4 Saffron

2.774×104

0.9011±0.089 0.003694

±3.2×103 Catechol

1.248×10

3.111×10-5±0.3 489

±0.0024 4

0.3185±0.015 4.848×10

±1.1×103 Pyrogallol

0.08329±0.031

297.2 ±7.5

-4

7.589× 10

-4

490 1.557×10 ±0.4 491 -5

±2.6×10-5

±6.3×10-5

0.02551

0.005049

5.176×10-4

492 7.981×10-6±2

±7.8×10-4

±8.2×10-5

±5.18×10-5

493 494

495

Standard error values were calculated from three measurements for each analyte by software itself

496

27



Page 28 of 29

497 498

Table 2 Changes in secondary structure of MT in presence of inhibitors

499 500 501 502 Inhibitors Wave length (nm) % changes

Pyrogallol 208 222

Phloroglucinol 208 222

Tannic acid 208 222

Saffron 208 222

Catechol 208 222

21.97 ±4.3

78.03 ±14.82

60.41 ±10.26

9.63 ±1.15

73.87 ±14.03

23.13 ±4.16

147 ±29.4

124.17 ±24.83

92.57 ±16.6

15.08 ±2.41

28


Page 29 of 29

503


TOC graphic

504

Tyrosinase

Tyrosinase

Screening using SPR Inhibitors

RU 180

130

R esponse

Binding sensogram

80

30

-20 -500

0

500

1000 Time

1500

2000

2500 s

505 506 507 508

29


Screening of Inhibitors for Mushroom Tyrosinase Using Surface

Recommend Documents