THI JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 249, No. 11, hue of June 10, PP. 2510-2618, 1974

Printed in U.S.A.

Photoaffinity Labeling of Peptide Hormone Binding Sites*

(Received for publication, November 27, 1973)

RICHARD E. GALARDY,$ LMMAN C. CRAIG, JAMES D. JAMIESON,~ AND MORTON P. PRINTZ~

From The Rockefeller University, New York, New York 10011

SUMMARY

The known binding of a derivative of the COOH-terminal tetrapeptide of gastrin with high biological activity to bovine serum albumin suggests a convenient model system for test- ing the affinity labeling of hormone binding sites. Photol- ysis of 4-azidobenzoylpentagastrin, Z-nitro-5-azidobenzoyl- pentagastrin, 4-acetylbenzoylpentagastrin, and 4-benzoyl- benzoylpentagastrin in the presence of bovine serum albumin attaches each peptide derivative covalently to the protein to the extent of 50 mole % using a 3-fold excess of peptide and moderate photolysis times. Under similar conditions using lysozyme for the protein, only 8 mole % peptide is bound. Oleic acid was found to compete for the binding sites on serum albumin. When serum albumin labeled with 4-azido- benzoylpentagastrin was split with cyanogen bromide most of the label was found on two of the peptides located in the middle of the albumin chain. A degree of specificity is thus indicated. These results suggest that the peptide derivatives should be capable of labeling their binding sites on tissue which is stimulated by the hormones gastrin and pancreozy . mm.

In addition, the model photoreactions of acetophenone and benzophenone with glycine were investigated and the photo- addition products isolated. The results of these model reactions and the successful labeling of albumin with ketone derivatives of pentagastrin suggest that ketones may be useful photoaffinity probes.

The use of photogenerated reagents for labeling biological ac- tive sites has been reviewed by Knowles (1). Photoaffinity probes have two main advantages over conventional active site- directed reagents. First, photoaffinity probes are inert until photolysis, permitting control experiments to be done before photolysis to insure labeling of the desired active site. In addi- tion, many photoaffinity probes can insert into carbon-hydrogen bonds. Thus, photoaffinity probes can label any binding site which contains carbon-hydrogen bonds and do not require the presence of particular reactive functional groups at the binding

* This investigation was sunnorted in part b.y National Insti- tutes of Health &ant AM 024%.

- -

i Present address. Section of Cell Bioloev. Yale Universitv Medical School, 333 Cedar Street, New Have;, Connecticut 06516.

0 Present address, Division of Pharmacology, Department of Medicine, University of California at San Diego, La Jolla, Cali- fornia 92037.

site. Specificity of labeling therefore depends solely on the spe- cific binding of ligand to receptor, which is then followed by a nonspecific covalent bond forming reaction that guarantees labeling of the binding site (1). Photoaffinity probes should be particularly useful for labeling antigen combining sites, regula- tory sites on enzymes, and hormone receptor sites, where reac- tive functional groups may not be present, but which surely con- tain carbon-hydrogen bonds.

The chemistry of aryl azide photoaffinity probes has been briefly discussed by Knowles (1). The photochemistry of aryl ketones is discussed in Turro (2). The reactive photogenerated triplet state of benzophenone has been suggested by Galardy et al. (3) to be a new photoaffinity probe, capable of bonding to many types of alkyl carbon atoms. Aryl ketones may have an advantage over other photoaffinity probes because the ketone triplet state is nearly inert to reaction with water.1 Thus, little ketone photoaffinity label will be lost to hydrolysis in a biological labeling experiment and the label need not be used in excess. Breslow and co-workers (5) and Baldwin et al. (6) have shown that photolysis of benzophenone derivatives attached to or com- plexed with other molecules containing methylene carbons fre- quently results in covalent bonding between the ketone carbonyl carbon and the attacked methylene carbon. Specificity of at- tack has resulted from forces orienting the ketone toward par- ticular methylene groups within the target molecule (5). Martyr and Benisek (7) have used ru,fi-unsaturated steroid ketones, which can undergo photoreactions similar to those of aryl ke- tones, to label and inactivate AK-3-ketosteroid isomerase.

Aryl azide and aryl ketone derivatives of the COOH-terminal gastrin pentapeptide were chosen as model compounds capable of affinity labeling binding sites for this hormone fragment. Peptides of this length are known to have high biological activity (8) and proved to be easily derivatized and purified. A deriva- tive of the gastrin tetrapeptide is known to bind to serum albumin at two equivalent binding sites with the modest association con- stant of 3 X lOa M-I (9, 10). The successful labeling of penta- gastrin binding sites on albumin as reported here implies that photoaffinity labeling using aryl azide or aryl ketone probes may be generally useful in the tagging and identification of peptide hormone binding proteins.

EXPERIMENTAL PROCEDURE

Materials

Crystallized bovine plasma albumin (Lot 572003) was from Armour. Formic acid (97 to loOyO), cyanogen bromide, and iodo-

1 For abstraction of hydrogen from water by benzophenone, k = lo* ~-1 s-1, and for abstraction of hydrogen from alcohols, k = 1W - 107 ~-1 s-r (4).

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TABLE I

Derivatives of benzoic acida

The abbreviations usedare: 4-Ns-BzOH. 4-azidobenzoic acid; ~-NOZ-~-N~-BZOH, 2-nitro-5-azidobenzoic acid; HONSu, N-hydroxysuc- cinimide; DME, 1,2-dimethoxyethane.

Compound

4-N3-BzOH

4-Nj-BzONSu

4-Ng-Bz-Gly

4-N3-Bz-Gly-ONSu

2-N02-5-N3-BzOH

2-N02-5-N3-BzONSu

2-N02-5-N3-Bz-Gly

2-N02-5-N3-Bz-Gly-ONSu

4-AC-BzONSu

4-AC-Bz-Gly

4-AC-Br-Gly-ONSu

4-Bz-BzOCI

4-Bz-Bz-Gly

4-Bz-Bz-Gly-OPhC15

MW

163.13

260.22

220.20

317.27

208.14

305.22

265.11

362.14

261.24

221.21

318.29

244.80

283.27

502.31

Analysis % tila .̂

C

51.54 51.23

50.77 51.06

49.09 48.72

49.21 49.16

40.39 40.33

43.29 43.19

40.74 40.46

43.10 42.87

59.77 59.57

59.73 59.64

56.60 56.67

68.73 68.81

67.84 67.94

49.70 49.65

H

3.09 3.29

3.09 3.22

3.67 3.76

3.49 3.75

1.96 1.94

2.31 2.47

2.66 2.85

2.78 2.90

4.24 4.37

5.01 4.95

4.63 4.43

3.71 3.84

4.63 4.78

2.28 2.32

N

25.76 26.00

21.53 21.55

25.44 25.62

22.08 21.81

26.92 27.12

22.95 22.78

26.42 26.26

23.29 23.60

5.36 5.36

6.33 6.30

8.80 8.80

4.95 4.93

2.64 2.54

Crystals from

H20:EtOH

dioxane:pet ether

MP

9

183

174-177

H20 137-139

Pr20H:dioxane 167-170

pet ether 165-166

CH2Cl2:pet ether

H20:EtOH

Pr20H:ether

Pr20H:OME

136-137

191-194 d

176-179 d

116-117

EtOH 192-194

DME:ether 176-180

cc14

EtOH

EtOAc

96-97

185-187

175-180

aAll derivatives had ir and uv spectra consistent with their structures.

acetic acid were from Matheson, Coleman, and Bell. lodoacetic acid was recrystallized from petroleum ether before use.

Organic Syntheses

Ac-Gly-OMeLGly-OMe-HCl (Sigma; 100 g, 0.8 mole), 250 ml of acetic anhydride, and 500 ml of tetrahydrofuran were stirred at room temperature during the slow addition of 300 ml of triethyl- amine. After 4 hours, the mixture was evaporated under vacuum and the residue distilled at 106112” at 0.5 mm. The distillate was recrystallized from methanol-ether to give 60 g (62%) of Ac-Gly- OMe, m.p. 53-55” (m.p. 58-59”, Dictionary of Organic Com- pounds).

4-Azidobenzoic Acid-To a stirred suspension of 13.7 g (0.1 mole) of 4-aminobenzoic acid (Sigma) in 150 ml of water and 20 ml of concentrated sulfuric acid cooled to -2” were added 8.5 g (0.12 mole) of sodium nitrite in 75 ml of water over a period of 20 min. The suspension dissolved. Urea was then carefully added with stirring to destroy excess nitrous acid. After 20 min, 13 g (0.2 mole) of sodium azide in 60 ml of water were added over a period of I5 min with continuous stirring. After evolution of nitrogen ceased, the mixt’lre was stirred for another 20 min and the solid product filtered off in the hood. The product was washed with ice water and recrystallized from ethanol and then from ethanol- water to give 10 g (6097,) of 4-azidobenzoic acid, m.p. 183” with de- composition (see Table I).

N-Hydroxysuccinimide Ester of 4.Azidobenzoic Acid-N-Hy- droxysuccinimide esters were prepared essentially according to Anderson et al. (11). To 4.9 g (30 mmoles) of 4-azidobenzoic acid and 3.45 g (32 mmole) of N-hydroxysuccinimide (Sigma) in 50 ml of dioxane cooled in ice were added 6.2 g (30 mmoles) of di- cyclohexylcarbodiimidz in 15 ml of dioxane. After 12 hours at 25” the dicyclohexyl urea was removed by filtration and the

* The abbreviation used is: Ac-Gly-OMe, N-acetylglycine methyl ester.

filtrate was evaporated to dryness. Recrystallization from di- oxane and then from dioxane-petroleum ether gave 2 g (3970) of the N-hydroxysuccinimide ester of 4-azidobenzoic acid, m.p. 174-177”.

&AzidobenzoyZglycine-To 2.5 g (30 mmoles) of sodium bicar- bonate and 1.1 g (15 mmoles) of glycine in 70 ml of water were added 3.9 g (15 mmoles) of the Nhydroxysuccinimide ester of 4- azidobenzoic acid in 140 ml of dioxane. After 12 hours the mixture was rotary evaporated to 40 ml, cooled in ice, and adjusted to pH 2 with concentrated hydrochloric acid. The solid product was collected and recrystallized from water to give 1.2 g (3670) of 4- azidobenzoylglycine, m.p. 137-139”.

N-Hydroxysuccinimide Ester of 4-Azidobenzoylglycine-To 0.88 g (4 mmoles) of 4.azidobenzoylglycine and 0.46 g (4 mmoles) of N-hydroxysuccinimide in 15 ml of dioxane at 0” was added 0.83 g (4 mmoles) of dicyclohexylcarbodiimide in 1 ml of dioxane. After 12 hours at 25”, the mixture was filtered and the filtrate evapo- rated. The residue was crystallized from isopropyl alcohol- dioxane to give 0.34 g (27%) of the N-hydroxysuccinimide ester of 4-azidobenzoylglycine. A small amount was recrystallized for analysis (m.p. 167-170”).

4-Azidobenzoyl-Gly-Trp-Met-Asp-Phe-NH2 (Q-Azidobenzoylpen- tagastrin-To 33 mg (100 pmoles) of the N-hydroxysuccinimide ester of 4.azidobenzoylglycine in 2.5 ml of dimethylformamide were added 10 mg (100 pmoles) of triethylamine and 70 mg (I00 pmoles) of Trp-Met-Asp-Phe-NH2 (tetragastrin, Schwarz-Mann). The suspension was stirred for 20 min until all the tetragastrin dissolved, left overnight, and then evaporated under high vacuum to near dryness. Countercurrent distribution in the system, chloroform-acetic acid-water (2: 1: 1)) in a BO-tube apparatus with 3 ml of each phase per tube, gave the pattern shown in Fig. la after 80 transfers. Tubes 9 to 21 were pooled and evaporated and the residue was lyophilized from 90% acetic acid to give 57 mg (65%) of peptide. Thin layer chromatography in 1-butanol-acetic acid-water (4: 1: 1) gave a single spot by Ehrlich’s reagent and by

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acid permanganate, RF 0.8. Electrophoresis at pH 5.6 gave a single spot staining by I’Zhrlich’s reagent but not by ninhydrin, which migrated to the anode. Amino acid analysis gave the cor- rect composition (see Table II).

4 - Azidobenzoyl- [2 - 3H]Gly - Trp -Met-Asp - Phe -NH, (4 - Azido- benzoyl-[3H]pentagastrirr)-One millicurie (33 nmoles) of [2-%- glycine (Schwarz-Mann) in 0.01 N hydrochloric acid was evapo- rated to dryness in small test tube. To the tube were added 390 pg (5 rmoles) of glycine, 250 hl of 2 M sodium bicarbonate, and 250 ~1 of the N-hydroxysuccinimide ester of 4-azidobenzoic acid in

dioxane. After 24 hours the reaction mixture was acidified to pH 2 with concentrated hydrochloric acid and extracted with 3 X 0.5 ml of ethyl acetate. The organic phase, which contained 87yo of the total counts, was dried over sodium sulfate, evaporated, and taken up in 100 ~1 of dioxane containing 575 pg (5 rmoles) of N-hydroxysuccinimide. To this were added 100~1 of dioxane con- taining 1.1 mg (5 pmoles) of dicyclohexylcarbodiimide. After 24 hours, the mixture was filtered and the filtrate was made up to 1.5 ml with pH 7.0, 2% sodium bicarbonate at O”, and extracted im- mediately with 3 X 0.5 ml of ethyl acetate. The organic phase, containing 90% of the total counts, was dried, evaporated, and taken up in 0.5 ml of dimethylformamide containing 3.5 mg (5 /rmoles) of tetragastrin and 500 rg (5 pmoles) of triethylamine. After 24 hours, the reaction mixture was evaporated to near dry- ness, and purified by countercurrent distribution as for 4-azido- benzoylpentagastrin, giving the pattern shown in Fig. lb. Tubes 9 to 22 were evaporated to near dryness, the mixture lyophilized from 90% acetic acid, and dissolved in 300 ~1 of dimethylsulfoxide for storage in the freezer. This material gave a single spot by Ehrlich’s reagent and by scanning for radioactivity on thin layer chromatography analysis. Amino acid analysis showed a yield of 3.3 I.rmoles (66%) of 4.azidobenzoyl-[SHJpentagastrin with the cor- rect amino acid composition.

%Nitrod-Azidobenzoyl-Gly-Trp-Met-Asp-Phe -NH, (%Nitro d- AzidobenzoyZpentagastrin)-2 -Nitro -5 - azidobenzoylpentagastrin was synthesized by the same route as 4-azidobenzoylpentagastrin starting with 2-nitro-5-aminobenzoic acid (Pfaltz and Bauer), except that all procedures were done in darkness or subdued light- ing. The analytical results for the intermediates leading to the N-hydroxysuccinimide ester of 2-nitro-5-azidobenzoylglycine are shown in Table I. The analytical results after the countercurrent distribution purification of 2-nitro-5-azidobenzoylpentagastrin (Fig. lc) are shown in Table II.

.2-Nifro&Azidobenzoyl- [aH]pentagastrin-2-Nitro-5-azidoben - zoyl-[3H]pentagastrin was synthesized by the same route as 4-azi- dobenzoyl-[3H]pentagastrin except that manipulation in the light was minimized. The analytical results after the countercur- rent distribution purification of 2-nitro-5-azidobenzoyl-[*H]penta- gsstrin (Fig. Id) are shown in Table II. The product was stored frozen in 300 ~1 of dimethylsulfoxide.

4-Acetylbenzoylpentagastrin-4-Acetylbenzoylpentagastrin was synthesized by the same route as 4-azidobenzoylpentagastrin, starting with 4-acetylbenzoic acid (Sapon). The analytical re- sults for the intermediates leading to the N-hydroxysuccinimide ester of 4-acetylbenzoylglycine are shown in Table I. The analyt- ical results after the countercurrent distribution purification of 4-acetylbenzoylpentagastrin are shown in Table II.

~-AcetylbenzoyZ-[aH]pentagas~~in-44Acetylbenzoyl- [aH]penta- gastrin was prepared by the same route as 4-acetylbenzoyl-[SH]- pentagastrin. The analytical results after countercurrent distri- bution purification of 4-acetylbenzoyl-]aH]pentagastrin are shown

(cl Z-NO*-5.N3- benzoylpentagastrln

(0) 4.N3-benzoylpentogastrln

0 20 40 60

( b) (d)

0 20 40 60 0 20 40 60

Tube number

FIG. 1. Countercurrent distribution of two of the substituted benzoylpentagastrins. The experimental details are given in the text. l , the theoretical curve in each case. a, 4-azidobenzoyl- pentagastrin; b, 4-azidobenzoyl-[aH]pentagastrin; c, 2-nitro-5- azidobenzoylpentagastrin; d, 2-nitro-5-azidobenzoyl-[aH]penta- gastrin. Countercurrent distribution of the two aryl ketone derivatives of pentagastrin gave patterns similar to those shown here.

TABLE II Synthetic peptides related to gastrin and pancreozymin

- Amino acid analysis

Yield from tetragastrin

specilic activityb Peptide, molecular weight RP

- As;cydtic Glycine Metbi-

onine

1.1 1.0 0.9 1.1 NDd 1.0 1.2 0.9 0.6 1.1 0.6 1.0 1.0 1.1 ND 1.1 1.1 0.8 1.1 1.0 1.0 1.2 0.9 1.0

Trypto- phaIl

1.0” 0.8 ND 0.8 l.OC 0.9 0.5” ND 1.0” 0.8 0.3” ND 0.8” 0.8 0.58 ND

% 65 66 63 55 83 35 63 42

CM/Mole

11

31

14

29

4-Azidobenzoylpentagastrin, 899. ................ 4-Azidobenzoyl-[3H]pentagastrin, 899 ............ 2-Nitro-5-azidobenzoylpentagastrin, 945. ........ 2-Nitro-5-azidobenzoyl-[3H]pentagastrin, 945. .... 4-Acetylbenzoylpentagastrin, 900 ................ 4-Acetylbenzoyl-[3H]pentagastrin, 900. ........... 4-Benzoylbenzoylpentagastrin, 962. .............. 4-Benzoylbenzoyl-[3H]pentagastrin, 962 ...........

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

- (1 Thin layer chromatography on Silica Gel G in butanol-acetic acid-water (4:l:l). b Counts per min under conditions described under “Methods,” not disintegrations per min. c Alkaline hydrolysis method described under “Methods.” d ND, not determined. 6 Acid hydrolysis method described under “Methods.”

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in Table II. The final product was dissolved in 300~1 of dimethyl- sulfoxide for storage in the freezer.

4-Benzoylbenzoylchloride-4-Bensoylbenzoic acid, 11.3 g (50 mmoles), (Aldrich) and 10.4 g (50 mmoles) of phosphorus penta- chloride were heated in a 100-ml flask fitted with a vacuum distill- ing head and condenser. Vacuum distillation was begun when the reaction mixture reached 160”. The product distilled at 196-198” at 9 mm, and was crystallized from carbon tetrachloride-n-heptane to give 9.1 g (75yo) of 4-benzoylbenzoylchloride. One gram of this material was recrystallized twice from CC14 for analysis, m.p. 95-96”.

4-Benzoylben.zoylgl~ci7le-4-Benzoylbenzoylglycine was prepared by the same procedure as 4-azidobenzoylglycine, except that 4- benzoylbenzoylchloride was used instead of the N-hydroxysuccini- mide ester.

Pentachlorophenylester of 4-Benzoylbenzoylglycine-The penta- chlorophenylester of 4-benzoylbenzoylglycine was prepared by the same method as the N-hydroxysuccinimide ester of 4-azidobenzo- ylglycine.

4 - Benzoylbenzoylpentagastrin - 4 - Benzoylbenzoylpentagastrin was prepared by the same method as 4.azidobenzoylpentagastrin using the pentachlorophenyl ester of 4-benzoylbenzoylglycine as the acylating agent. The analytical results after the counter- current distribution purification are shown in Table II.

4-BenzoylbenzoyZ-[JH]pentagastrin-4-Benzoyl-~3H]pentagastrin was prepared by the same method as 4-azidobenzoyl-[3H]penta- gastrin, except that the initial acylating agent was 4-benzoyl- benzoylchloride. The analytic&l results after the countercurrent distribution purification are shown in Table II. The final product was dissolved in 300 ~1 of dimethylsulfoxide for storage in the freezer.

Methods

Analytical Melhods-Melting points were taken on a heated microscope stage and are corrected. Infrared spectra were re- corded on a Perkin-Elmer 237 grating spectrophotometer. Ultra- violet spectra were recorded on a Cary model 14 spectrophotom- eter. Absorbance at any specific wavelength was measured on a Beckman DU monochromator equipped with a Gilford No. 222 photometer. Thin layer chromatography was on glass plates coated with Silica Gel G (Brinkmann) according to Randerath (12). Detection was with Ehrlich’s reagent according to Stewart and Young (13), with acid permanganate according to Randerath (12), or by spraying with 2% ninhydrin in acetone. Plates were scanned for radioactivity using a Varian Aerograph plate scanner. High voltage paper electrophoresis was in a flat bed Pherograph apparatus at 0” on MN paper No. 214 (Brinkmann) at approxi- mately 60 volts per cm. Peptides were visualized with Ehrlich’s reagent or ninhydrin as in thin layer chromatography. Sodium dodecyl sulfate gel electrophoresis was according to Weber and Osborn (14) except that the acrylamide stock solution was made up with 1.6% instead of 0.6% methylenebisacrylamide for separat- ing the alanine and aspartic acid cyanogen bromide peptides of bovine serum albumin. Alkaline hydrolyses and manual ninhy- drin analyses were according to Moore and Stein (15) and Fruchter and Crestfield (16) with ninhydrin in dimethylsulfoxide (17). Amino acid analyses were made on a Beckman model MS instru- ment,. For determining methionine or tryptophan in acid hy- drolyses, 1 drop of mercaptoacetic acid was added to the hydroly- sis tube (18). Tryptophan was also determined by alkaline hydrolysis (19). Scintillation counting was with a Packard Tri-Carb instrument, in 5 ml of Protosol (New England Nuclear).

Photolysis-The photolysis apparatus consisted of a Hanovia 450-watt high pressure mercury immersion lamp in a water- jacketed borosilicate immersion well. Reaction vessels were flushed with nitrogen for 15 min before photolysis and then clamped against the well with vessel and well enclosed together in aluminum foil. Photolyses in the presence of amino acids, pep- tides, or proteins were done at 4’ by immersing well and reaction vessel in a water bath cooled by circulating 50yo ethanol kept at -15” through a coil in the water bath. For solutions containing proteins, the buffer was deoxygenated by bubbling nitrogen through it before the addition of the protein as a solid or as a concentrated solution. Photolysis without additional filters occurred with wavelengths 2280 nm being transmitted through the Pyrex well and vessel. A Corning No. 3220 immersion filter was used for photolyses with light of wavelengths 2320 nm.

Photolysis of Acelophenone and Ac-Gly-OMe in Benzene-Aceto- phenone (2 g, 16 mmoles) and 20 g (150 mmoles) Ac-Gly-OMe were dissolved in 600 ml of distilled benzene. After 55 hours of irradi- ation at ambient temperature at wavelengths 2280 nm, only one- half the original acetophenone remained as determined by ultra- violet analysis. Rotary evaporation of the benzene gave a residue which was taken up in chloroform and then washed with water. The material in the chloroform layer was subjected to counter- current distribution in the system, chloroform-water-methanol (2: 1: 1), in a lOO-tube machine with 10 ml of each phase per tube. Two ninhydrin-positive bands were found by alkaline hydrolysis of aliquots of each tube. The fast moving ninhydrin-positive band had no ultraviolet absorption at 260 nm and was Ac-Gly-OMe. The slow moving band, with absorption at 260 nm, was evaporated under vacuum, taken up in 3 ml of chloroform-cyclohexane (1: l), and chromatographed on grade III neutral alumina (25 X 0.9 cm) (Woelm). Elution with chloroform-cyclohexane yielded a fraction containing mostly acetophenone by its infrared and NMR spectra. Further elution with chloroform gave a fraction which crystallized from chloroform-cyclohexane to give 169 mg (8%) of the diastereomers of Compound 1, Fig. 2, by NMR. Recrystalliza- tion from ethyl acetate gave 66 mg of one of the diastereomers of 1, m.p. 150-151”; NMR (Varian HR 220, CDCIS): 61.61 (s, 3H), 1.89 (s, 3H), 3.32 (s, lH), 3.76 (s, 3H), 4.86 (d, lH, J = 9), 6.23 (d, lH, J = 9), 7.42 (multiplet, 5H); IR (CHCla) : 1673, 1728, 3420 cm-l.

C&n~NO,

Elemental analysis: C 61.86, H 6.70, N 5.45 Calculated: C 62.14, H 6.82, N 5.57

A ninhydrin-negative band with ultraviolet absorption at 260 nm preceded the slow moving ninhydrin-positive band in the countercurrent distribution pattern. This band was evaporated and chromatographed in the same manner as the ninhydrin-posi- tive band. Elution with chloroform-cyclohexane (1:1) gave a fraction containing mostly acetophenone by its NMR and IR spectra. Elution with chloroform gave two fractions, each of which were evaporated and then crystallized from chloroform- cyclohexane. The first fraction gave 110 mg of material with a broad melting range. The second fraction gave 130 mg of the pinacol 2 of Fig. 2, m.p. 124-125”, possibly dl-2,3-diphenyl-2,3- butanediol by its melt,ing point (dl-2,3-diphenyl-2,3-butanediol, m.p. 12&126”, meso-2,3-diphenyl-2,3-butanediol, m.p. 120-121” (20)). NMR (Varian A 60, CDCl3): 61.68 (s, 6H), 2.76 (s, 2H), 7.17 (multiplet, 10H).

Photolysis of Acetophenone and Ac-Gly-OMe in Water-Aceto- phenone (1 g, 8.3 mmoles) in 50 ml of 50y0 w/v Ac-Gly-OMe (190 mmoles) in water was photolyzed for 116 hours at wavelengths 2280 nm, until only 20% of the original acetophenone remained by ultraviolet analysis. The photolysis mixture was extracted with chloroform and the extract subjected to countercurrent distribution as described above. The band containing Compound 1 was chromatographed as described above and crystallized from chloroform-cyclohexane to give 89 mg (6%) of a mixture of the diastereomers of 1 by NMR. Recrystallization from chloroform- cyclohexane and then from ethyl acetate gave one of the diastere- omers of 1, m.p. 150-151”. This compound was shown to be identi-

OH OH

3 4

FIG. 2. The photoaddition of acetophenone (a) and benzophe- none (b) to Ac-Gly-OMe.

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cal with compound 1 isolated from the photolysis in benzene by comnarison of Ilt and NMR spectra and by mixed m.p. 149-150”.

Piololysis of Benzophenone and Ac-Gly-OMe in Benzene-The isolation of Compound 9, Fig. 2, from the photolysis of benzo- phenone and Ac-Gly-OMe in benzene with radiation of wavelength 2320 nm has been described by Galardy et al. (3). Compound 3 was isolated in 30% yield and gave the following analysis:

C,sH,,NO4

Elemental analysis: C 69.07, H 603, N 4.42 Calculated: C 69.00, H 6.11, N 4.47

Photolysis of Benzophenone and Ac-Gly-OMe in Waler-The iso- lation of Compound 3 from the photolysis of benzophenone and Ac-Gly-OMe in water has been described by Galardy et al. (3). Compound 3 was isolated in 18% yield and was shown to be identi- cal with Compound 3 isolated from the photolysis in benzene by comparison of NM11 and IR spectra and by mixed melting point,

Photodestruction of Free Amino Acids and of Serum Albumin-A standard mixture (Beckman) of equimolar amounts of amino acids diluted to lo-’ M in 0.1 M NazHPOa or 0.1 M NaHzC03 buffer at pH 7.4 was photolyzed under nitrogen at 4” for 5 hours at wave- lengths 2280 nm or for 2 hours at wavelengths 2320 nm, and then analyzed for remaining amino acids. Where the losses of amino acids were small, the photolyses were continued and analyses were performed at time intervals in order t,o establish the first order loss more accurately. The photolyses were then repeated in the presence of 4-acetylbenzpic acid (at wavelengths 2280 nm) and (at wavelengths 2320 nm) 4-azidobenzoic acid, 2-nitro-5-azido- benzoic acid, and 4-benzoylbenzoic acid present at the same con- centration as a single amino acid in the mixture (10T4 M) and fol- lowed by amino acid analyses.

The photodestruction of serum albumin alone at lo+ M in neu- tral buffer was measured using the same methods employed for the free amino acids, except that appropriate hydrolysis preceded amino acid analysis. Samples of albumin photoaffinity labeled by 4-acetylbenzoylpentagastrin, 4-benzoylbenzoylpentagastrin, 4-azidobenzoylpentagastrin, and 2-nitro&azidobenzoylpenta- gastrin were subjected to amino acid analysis in order to determine the photodestruction of amino acid residues in a model affinity labeling experiment.

Photoafinity Labeling of Pentagastrin Binding Sites on Serum Albumin-Bovine serum albumin at a concentration of about 0.1 rnM in neutral buffer was incubated for 30min at room temperature or at 37” with approximately a 2- or a-fold excess of each of the radioactive pentagastrin derivatives, before photolysis, at 4” in Pyrex test tubes sealed with screw caps. After photolysis, the mixture was separated on Sephadex G-25 and the pentagastrin eluted with the albumin peak was determined. The amount of peptide attached to albumin after photolysis was not very sensi- tive to small changes in the ratio of peptide to protein in the photolysis mixture. The albumin peak was occasionally hydro- lyzed for amino acid analysis to check the recovery of albumin. Photolysis of 4-azidobenzoylpentagastrin and 4-acetylbenzoyl- pentagastrin was for approximately two half-lives of the photo- affinity label chromophore. Photolysis of 2-nitro&azidobenzoyl- pentagastrin and 4-benzoylbenzoylpentagastrin was for at least five half-lives of the photoaffinity label chromophore. The details of a representative labeling experiment with each of the four pentapeptides are shown in Table III.

For each of the four pentagastrin derivatives, albumin was sepa- rated from free peptide on Sephadex G-25 in 1% NH,HCO, without previous photolysis to show that strong binding occurred only after photolysis. For 4-acetylbenzoylpentagastrin, as shown in Fig. 3a, 0.10 rmole of tritiated peptide (1.6 X lo6 cpm per rmole and 0.04 rmole of albumin in 0.2 ml of 1% NH,HCOp were incu- bated for 30 min at 37” and then chromatographed. In addition, for each of the four pentagastrin derivatives, labeled albumin was separated from free peptide on Sephadex G-25 in 1% NHaHCO$ buffer containing 0.1% sodium dodecyl sulfate to show that bind- ing after photolysis was strong enough to suggest the formation of a covalent bond. The separation of 2-nitro-5-azidobenzoyl- pentagastrin-labeled albumin from free peptide in the presence of sodium dodecyl sulfate is shown in Fig. 3b. The photolysis conditions were those given for 2-nitro-5-azidobenzoylpenta- gastrin in Table III.

Two additional experiments were done in order to determine the

(0) Wlthout photolysls 05 AlbumIn

04 ! I

cpm per 0.3ml

2 Ic) With oleate (d) With lysozyme

- cpm per 0.5r-n

0 J IO 20 30

ml

(b) Chromatography In SDS I

cpm per 0. I ml

3000

2000

cpm per 05llll

ml

FIG. 3. Separation of photoaffinity labeled protein from free pentagastrin on Sephadex G-25. The experimental details are given in the text. V, void volume of the column in each case. a, separation of 4-acetylbenzoylpentagastrin from serum albumin without previous photolysis; b, separation of 2-nitro-5-azidoben- zoylpentagastrin-labeled albumin from free pentapeptide in the presence of sodium,dodecyl sulfate; c, separation of 4-acetylben- zoylpentagastrin from serum albumin labeled in the presence of sodium oleate; d, separation of 4-acetylbenzoylpentagastrin-la- beled lysozyme from free pentapeptide.

TABLE III Photoafinity labeling of bovine serum albumin at 4"

Peptide (rmole) Specific activity BUtk Peptide

attached to albumin

cpn/~mole ~molc hrs mole qo

4-Azidobenzoylpentagastrin (1.5). 5.5 x 106 0.7 3.2mlO.l~Tris-HCl,pH7.4 2 49

2-Nitro-5-azidobenzoylpentagastrin (0.3). 3.6 X lo8 0.095 1.3 ml 1% NH*HCO, 0.5 60

4-Acetylbenzoylpentagastrin (0.52). 7.3 x 106 0.31 2 ml 1% NHdHC03 8 48

4-Benzoylbenzoylpentagastrin (0.3). 3.4 x 108 0.095 1.3 ml 1% NHIHCOa 2 41

0 These specific activities resulted from diluting the appropriate derivative of (2-aH]Gly-Trp-Met-Asp-Phe-NHt with the correspond- ing nontritiated peptide.

6 A Corning No. 3220 filter was used in all photolyses except that of 4-acetylbenzoylpentagastrin.

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10) CNBr fragments of albumln cpm per

(b) Rechromatogrophed C

0 160 320 460 640 SO0

ml

FIG. 4. Chromatography of the two unreduced cyanogen bromide cleavage fragments of photoaffinity labeled. bovine serum albumin on Sephadex G-75 (203 X 2.6 cm) in 0.1 M NaCl, 5% pro- panoic acid. a, initial separation of the N and C fragments; 1.2 #moles of N were isolated, as determined by amino acid analysis; b, rechromatography of Fragment C gave a recovery of 1.3 pmoles of C, as determined by amino acid analysis.

nature of the pentagastrin binding sites on albumin and to de- termine the snecificits of labeling. Tritiated 4-acetylbenzoyl- pentagastrin (6.133 pm&e, 1.4 X lO:cpm per pmole) and-O.03 pmble of albumin in 0.2 ml of 1% NH~HCOI containinn 0.5 umole of so- dium oleate were photolyzed for8 hours without rhe Corning filter, and then chromatographed, as shown in Fig. 3c. In addition, 0.133 pmole of tritiated 4-acetylbenzoylpentagastrin (1.4 X lo6 cpm per pmole) and 0.04 pmole of lysozyme in 0.2 ml of l?& NHdHCOs were photolyzed for 8 hours without the Corning filter and then chromatographed, as shown in Fig. 3d.

Location of Pentaaaslrin Bindino Sites on Serum Albumin-To 340 mg (5 &oles) 0: half-cystinyl”bovine serum albumin (21) in 35 ml of pH 7.9 0.1 M Tris-HCl were added 13.3 mg (15 rmole) of tritiated 4-azidobenzoylpentagastrin of a specific activity of 3.82 X 10’ cpm per pmole. The solution was photolyzed under nitrogen (the buffer had been flushed with nitrogen before the addition of albumin) at wavelengths 1320 nm (with the Corning filter) for 6 hours at 4’. The solution was concentrated to 5 ml by ultrafiltration and chromatographed on Sephadex G-25 (55 X 2.8 cm) eluted with 1% NHIHCOS. The albumin peak carried 1 amole of pentagastrin per pmole of albumin. The albumin frac- tion was ultrafiltered to 20m1, dialyzed against 3 X 4 liters of water over 24 hours, and treated with cyanogen bromide in formic acid according to King and Spencer (21). The two resulting compo- nents, Fragment N and Fragment C, which contain peptides cross- linked by &sulfide bonds (see Fig. 8), were separated on a column of Senhadex G-75 (203 X 2.6 cm) eluted with 0.1 M NaCl. 5% propanoic acid, according to McMenamy and Weslowski ‘(22): Fragment C was concentrated by ultrafiltration and then rechro- matographed on the same column (Fig. 4b).

Fragment C was again concentrated by ultrafiltration and then reduced and carboxymethylated according to King and Spencer (21). The arginyl, prolyl, and glutamyl cyanogen bromide pep- tides of Fragment C (see Fig. 8) were separated on Sephadex G-150 (200 X 0.9 cm) in 1% NHdHCOa (21), lyophilized, and then sepa-

(0) Fragment C 40

A Arg pepllde

I200

800

400

. 2e 56 64 112 ml 3 1

(b) Rechmmatographed C Peptides g - 1

7r A Arg pepllde

32 64 96 I28

ml FIG. 5. a, chromatography of the reduced and carboxymethyl-

ated cyanogen bromide peptides of photoaffinity-labeled Fragment C on Sephadex G-150 (200 X 0.9 cm) in 1% NHdHCOa. b, a com- posite elution profile of the individually rechromatographed arginyl (circles), prolyl (squares), and glutamyl peptides (tri- angles). The inset shows the bands resulting from the arginyl and prolyl peptides on sodium dodecyl sulfate gel electrophoresis. Recoveries from the chromatography were 400 nmoles of arginyl, 250 nmoles of prolyl, and 335 nmoles of glutamyl peptides, as de- termined by amino acid analysis.

rately rechromatographed on the same column (Fig. 5). Frag- ment N was concentrated by ultrafiltration and reduced and carboxymethylat’ed (21). The alanyl and aspartyl peptides of Fragment N were chromatographed on Sephadex G-100 (400 X 0.9 cm) in 1% NHdHCOa (21) but were not well resolved (Fig. 6~). The two cuts shown in Fig. 6a, each enriched in the alanyl and aspartyl peptides, respectively, were rechromatographed on the same column (Fig. 6, b and c). Cuts from each of these chromato- grams, as shown in Fig. 6, b and c, were further enriched in their respective alanyl and aspartyl peptides, but were still cross-con- taminated and were chromatographed a third time.

RESULTS

Photodestruction of Free Amino Acids and of Serum Albumin- In order to determine the effects of the photolysis conditions on proteins during the photoaffinity labeling reaction, both free amino acids and serum albumin were examined for damage after photolysis. For all sulfur-containing amino acids in solution, the conditions necessary for rapid photolysis of acetophenone (wavelengths 2280 nm) were found to be severely damaging, in that 45% of the cysteine and 30% of the methionine were de- destroyed. Under the same conditions about 3yo of the tyrosine and 3% of the tryptophan were also destroyed. In the presence of 4-acetylbenzoic acid, more destruction occurred with loss of 80% of the cysteine and 60% of the methionine. Photolysis of serum albumin under these conditions caused less photodestruc- tion of amino acid residues with only destruction of cysteine (40%) (total cysteine contained as cysteine and cystine measured as cysteic acid) in albumin alone but with the additional loss of

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- (o) Fragment N

cpm per 05ml

2or

0 I5 30 45 60 75 90 I05

ml

i bi Ala pepilde (cl Asp peptlde ‘~“O,“‘,‘I

160

SO

40

16 39 52 65 78 91

ml

FIG. 6. a, chromatography of the reduced and carboxymethyl- ated cyanogen bromide peptides of Fragment N on Sephadex G-100 (200 X 0.9 cm) in 1% NH,HCO,. b, rechromatography of the alanyl peptide (Cut 1 in a). c, rechromatography of the aspartyl peptide (CUL 2 in a).

40% of the methionine and 5% of the tyrosine, phenylalanine, and tryptophan in albumin in the presence of 4-acetylbenzoyl- pentagastrin. These losses of the sulfur-containing amino acids are unacceptable and acetophenone-like photoaffinity probes could only be useful where the postphotolysis condition of the protein being labeled is not important.

The photolysis conditions for benzophenone (wavelengths 2320 nm) were found to be relatively nondestructive. Photol-

ysis of free amino acids or of serum albumin for 2 hours under these conditions caused no detectable loss of any amino acids. However, photolysis of free amino acids in the presence of 4- benzoylbenzoic acid caused the destruction of 457, of the cys- teine, 90% of the methionine, and 20% of the tyrosine and tryptophan present. In contrast, in an actual photoaffinity labeling experiment with serum albumin, no detectable loss of any amino acids occurred during labeling of the protein with 4- benzoylbenzoylpentagastrin. No losses of either free amino acids or amino acid residues in albumin were found in the pres- ence of the aryl azide labels.

Covalent Labeling of Pentagastrin Binding Sites on Serum Al- bumin-Four pentagastrin derivatives were tested at a concen- tration of about 0.3 mM for their ability to photoaffinity label 0.1 mM bovine serum albumin. The results are shown in Table III. In other experiments using a 5- to IO-fold excess of peptide to protein for prolonged photolysis times, up to 2 moles of both 4- azidobenzoylpentagastrin and 4-acetylbenzoylpentagastrin could be attached per mole of albumin, in agreement with the two binding sites thought to be present on albumin for a fluorescent derivative of tetragastrin (9, 10). The other two peptides were not tested for their ability to heavily label albumin. Human

serum albumin was similarly labeled by the peptides under both moderate and prolonged photolysis conditions.

Fig. 3 shows the separation of free pentapeptide from bovine serum albumin on Sephadex G-25 eluted with 1 y0 ammonium bi- carbonate in four representative experiments. In a, no 4- acetylbenzoylpentagastrin was attached to albumin in the ab- sence of photolysis. In b, about 60 mole y0 2-nitro-5-azido- benzoylpentagastrin was found attached to albumin after chro- matography in 1 y0 ammonium bicarbonate in the presence (Fig. 3b) or absence (see results in Table III) of 0.1 y0 sodium dodecyl sulfate. In c, 16 mole y0 4-acetylbenzoylpentagastrin was found attached to albumin when the photolysis was done in the presence of a 5-fold excess of sodium oleate, compared to 48 mole % attached when sodium oleate was not present (Table III). Oleic acid binds strongly to albumin (23) and should compete for hydrophobic binding sites on the protein3 In Fig. 3d, 8 mole y0 4-acetylbenzoylpentagastrin was found attached to ly- sozyme under conditions where 48 mole 7O was found attached to albumin (Table III).

Location of Pentagastrin Binding Sites on Serum Albumin- Fig. 4 shows the separation on Sephadex G-75 of the N and C fragments (containing peptides cross-linked by disulfide bonds) of bovine serum albumin photoaffinity labeled with 4-azidoben- zoylpentagastrin. The albumin carried 1.0 pmole of 4-azidoben- zoylpentagastrin per pmole of protein. Fragment N of Fig. 4a and rechromatographed Fragment C of Fig. 4b were found to be homogeneous by amino acid analysis and carried 0.42 pmole and 0.58 pmole of the pentapeptide per pmole of N and C respectively.

Fig. 5a shows the separation of the reduced and carboxy- methylated cyanogen bromide peptides of 4-azidobenzoylpenta- gastrinlabeled Fragment C on Sephadex G-150. The arginyl, prolyl, and glutamyl peptides of Fragment C shown in Fig. 5a were individually rechromatographed on the same column, as shown in Fig. 5b. Each peptide was found to be homogeneous by amino acid analysis and the arginyl and prolyl peptides were single bands on sodium dodecyl sulfate gel electrophoresis as shown in the inset to Fig. 5b. The glutamyl peptide was not subjected to electrophoresis since it is known not to stain with Coomassie blue dye.3 The arginyl, prolyl, and glutamyl pep- tides were found to be carrying 0.23, 0.06, and 0.01 pmole of 4-azidobenzoylpentagastrin per pmole of peptide, respectively. The 0.30 I.tmole of pentagastrin found on these three peptides is significantly less than the 0.58 pmole of pentagastrin originally found on Fragment C, which is known to contain only the arginyl, prolyl and glutamyl peptides, cross-linked by disulfide bonds.

Fig. 6a shows the chromatography of the reduced and car- boxymethylated cyanogen bromide peptides of 4-azidobenzoyl- pentagastrin-labeled Fragment N on Sephadex G-100. Cut 1, the alanyl peptide, appears to be carrying most of the tritium label, and was rechromatographed on the same column, as shown in Fig. 6b. Although Fig. 6b appears to show a single peak, sodium dodecyl sulfate gel electrophoresis of Cuts 1 and 2 of Fig. 6b show some contamination by aspartyl peptide (see the two left hand gels in Fig. 7a). Rechromatography of Cut 2, Fig. 6a, on the same cclumn also appeared to show a single peak corresponding to the aspartyl peptide, as shown in Fig. 6c. How- ever, sodium dodecyl sulfate gel electrophoresis of Cuts 1 and 2 of Fig. 6c showed contamination by alanyl peptide (see the two right hand gels of Fig. 7a).

In order to quantitate the amount of tritium label carried

3 Dr. T. P. King, Rockefeller University, personal communica- tion.

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1 and 9) demonstrates the ability of ketone photoaffinity probes to covalently bond to amino acid residues. The covalent struc- tures assigned to these new compounds are in agreement with the experimentally observed elemental analyses and the IR and NMR spectra. Both compounds were isolated as colorless high melting solids. Only one diastereomeric pair of Compound 1 was isolated and its absolute optical configuration was not deter- mined. The use of the substrate Ac-Gly-OMe as a model for the site of attachment of a ketone photoaffinity probe was sug- gested by the work of Elad and Sperling and their collaborators (24, 25, and references contained therein). Our results indicate that ketone photoaffinity probes should add to the ar carbon of glycine in proteins as well as adding to secondary and tertiary carbons in amino acid side chains by analogy with known photo- additions to such substituted carbons (5,6). The ketone triplet state may, however, have some preference for glycine a! hydrogens in proteins by analogy with the work done by Sperling and Elad (24). Since similar yields of addition products were isolated from the photoreactions in benzene and in water solution for each ketone, aryl ketone photoaffinity probes should be capable of labeling binding sites both on the hydrophilic surface and within the hydrophobic interior of a globular protein. The relative inertness of the ketone triplet state to reaction with water (4) should guarantee that no ketone photoaffinity label will be lost to reaction with water, in contrast to carbene photoaffinity labels, where immediate or delayed hydrolysis has frequently been ob- served to be the major reaction (26-28).

The photolysis conditions employed here for benzophenone and the aside chromophores damaged neither free amino acids nor bovine serum albumin at 4”. Free sulfur-containing amino acids were significantly destroyed by photolysis in the pres- ence of benzophenone, although no amino acid residues in serum albumin were lost under identical conditions. Although the experimental conditions for using acetophenone-like photo- affinity probes were found to be too damaging to sulfur con- taining and aromatic amino acids, benzophenone and aryl azide photoaffinity labeling experiments should leave most proteins intact except for the possible loss of sulfur-containing amino acids in some cases with benzophenone.

The results of labeling pentagastrin binding sites on bovine serum albumin with the aryl aside and aryl ketone derivatives of pentagastrin demonstrate the potential for labeling peptide hormone binding proteins in tissue with these peptides. The competition of oleic acid for the sites labeled in albumin sug- gests that the pentagastrin peptides are attaching to a general hydrophobic binding site on the protein. The low level of attachment of 4-acetylbenzoylpentagastrin to lysozyme, when compared to the attachment to albumin under identical condi- tions, further suggests that a genuine binding site is being la- beled on albumin. Although lysozyme is known to bind a variety of ionic and hydrophobic ligands (29), the extent of labeling of lysozyme by 4-acetylbenzoylpentagastrin was only 16% of that seen with albumin.

The location of the labeling of albumin by 4-azidobenzoyl- pentagastrin was not unique and showed a distribution of la- beling with the maximum near the middle of the primary struc- ture of the protein. The lack of highly specific labeling in this case is probably due to the low affinity of gastrin peptides of this length for their binding sites on albumin (K = 3 x 10” I++) or to the presence of more than one binding site, or both (10).

FIN. 7. Sodium dodecyl sulfate gel electrophoresis of the alanyl and aspartyl cyanogen bromide peptides of albumin. a, from left to right, alanyl Cut 1, Fig. 66, alanyl Cut 8, Fig. 6b, aspartyl Cut 1, Fig. 6c, aspartyl Cut 8, Fig. 6c. b, alanyl Cut 1, Fig. 6b, subjected to co-electrophoresis with genuine alanyl peptide, which was a gift of Dr. T. P. King of The Rockefeller University.

+-Fragment N ---t--- Fragment C F

FIG. 6. Schematic diagram of the primary structure of bovine serum albumin (21). The distribution of tritium label in albumin labeled with 4-aeidobensoylpentagastrin is given for each of the five cyanogen bromide peptides. The per cent label found on each peptide was calculated by taking the total label finally found on the five peptides (0.55pmole of label per eole of albumin) as 160%.

by the alanyl and aspartyl peptides, duplicate gels of Cut 1, Fig. 6b (alanyl peptide), and Cut b, Fig. 6c (aspartyl peptide), were run. The bands corresponding to the alanyl and aspartyl peptides were cut out by hand, hydrolysed for amino acid anal- ysis, and counted for tritium. The alanyl and aspartyl peptide bands were not cross-contaminated as determined by amino acid analysis. The alanyl peptide carried 9 times the amount of tritium per mole as the aspartyl peptide. To further establish the amount of labeling, Cut 1 of Fig. 6b (alanyl peptide) and Cut 8 of Fig. 6c (aspartyl peptide) were individually rechro- matographed on Sephadex G-100, (400 X 0.9 cm), eluted with 1% NHdHCOa. Each chromatogram showed a single peak about 80% homogeneous by amino acid analysis and sodium dodecyl sulfate gel electrophoresis. The alanyl peptide carried 0.22 pmole and the aspartyl peptide carried 0.027 pmole of 4-azidobenzoylpentagastrin per pmole of peptide. Again, a discrepancy exists between the 0.42 pmole of pentagastrin found per pmole of Fragment N before disulfide reduction and the 0.25 pmole of pentagastrin found on the alanyl and aspartyl peptides resulting from the reduction of Fragment N. These results are summarized in Fig. 8, which shows the schematic primary structure of bovine serum albumin given by King and Spencer (21). The percentage of tritiated 4-azidobenzoyl- pentagastrin found on each of the cyanogen bromide peptides is indicated.

DISCUSSION

The isolation of the photoaddition products of both aceto- phenone and benzophenone with Ac-Gly-OMe (Fig. 2, Structures

The discrepancy between the absolute amounts of 4-azido- benzoylpentagastrin found on Fragments C and N and the amounts finally found on the five cyanogen bromide peptides is

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not explicable at this time. Possibly the covalent bond linking the pentagastrin and its binding site, whose nature was not determined in these experiments, is sensitive to one of the rea- gents present during the reduction and carboxymethylation of Fragments N and C. The covalent bond expected when affinity labeling with aryl nitrenes is the N-C bond of the substituted

aniline PhNH-G- (l), which should be stable to all of the

procedures emplobed in isolating the cyanogen bromide peptides of albumin. However, the addition of the nitrene intermediate to a nucleophile can occur (l), and the product, a substituted azepine, may be sensitive to attack by sulfhydryl nucleophiles.’ The results of the labeling are, however, encouraging and are the first example of the photoaffinity labeling of peptide hor- mone binding sites.

Two of the pentagastrin derivatives, 4-azidobenzoylpenta- gastrin and 2-nitro-5-azidobenzoylpentagastrin, have been used in additional experiments to label binding sites for these peptides on the exocrine cells of the guinea pig pancreas. The results of these experiments will be reported in a separate publication.

Acknowledgment-The authors thank Dr. T. P. King of The Rockefeller University for his criticism and advice during the photoaffinity labeling experiments with serum albumin.

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versity

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Richard E. Galardy, Lyman C. Craig, James D. Jamieson and Morton P. PrintzPhotoaffinity Labeling of Peptide Hormone Binding Sites

1974, 249:3510-3518.J. Biol. Chem. 

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