2
80
V. AZARIAN ET AL.
MALDI-TOF MS, including a control sample of pure trypsin. The resulting
mass was compared to previously predicted values of fragment mass, based on
the hydrolytic reactivity of trypsin towards GVIA (Figure 2).
Calculating the specific activity (mAU/nmol) of K24-FGVIA
Specific activity was determined by a chromogenic protein assay, exclusive for
1
2
peptides containing hydroxyproline residues. First, a series of standard
solutions containing 0–5 mg of GVIA (in increments of 0.5 mg) were prepared
in water. Each solution was dried under reduced pressure, reconstituted in
6
00 ml of 6 M HCl, and transferred to individual test tubes. The tubes were
sealed and refluxed at 1108C for 16 h to ensure complete hydrolysis, and dried
under reduced pressure. Second, the assay buffer was prepared containing
anhydrous citric acid (4.57 g, 23.8 mmol), 1.2 ml glacial acetic acid, sodium
acetate trihydrate (12.0 g, 146 mmol), sodium hydroxide (3.4 g, 85.0 mmol),
and 100 ml toluene in deionized water (1 l). Each tube of dried hydrosylate was
reconstituted in 2 ml of assay buffer. The standards were then treated with 1 ml
of chloramine-T reagent (1.41 g chloramine-T, 26 ml n-propanol, and 53.3 ml
assay buffer per 100 ml of solution), and allowed to stand at room temperature
for 20 min. A 1 ml aliquot of a solution containing p-dimethylaminobenzalde-
hyde (15 g, 101 mmol), n-propanol (60 ml), and 26 ml perchloric acid (40%)
was added to each tube. The tubes were placed in a water bath at 608C for
1
5 min, and immediately transferred to a new water bath at room temperature
for 5 min. Absorption measurements were made at 550 nm for each standard
solution, and a curve was formulated. To correlate this standard curve with
absorption values at 215 nm for K24-FGVIA, the conjugate was purified by
RP-HPLC, the peak area at 215 nm (mAU ꢀ Sec) recorded, and the
hydroxyproline assay applied. The resulting molar amount of K24-FGVIA,
as determined by the Hyp-assay, was divided by the absorption area at 215 nm
to yield a conversion factor between area and mass quantity.
1
8
Preparation of K[ F]F-K complex
222
1
8
18 18
F] fluoride ion, in deionized water, was produced using the O(p,n) F
[
18
14
reaction by bombarding [ O]H O in a low volume (300 ml) silver target. The
2
activity, ranging from 5.6 to 6.7 GBq, was transferred to a solution containing
3
0 ml of 0.2 M K CO and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-
2
3
hexacosane (4.5 mg, 6 nmol) in 50 ml of acetonitrile. Water was removed via
azeotropic evaporation (4 ꢀ 200 ml) of anhydrous acetonitrile under an argon
stream at 1508C.
18
18
Preparation of 4-[ F]fluorobenzoic acid ([ F]FBA, 3)
The preparation of 3 was modified from the protocols described by Lee and
7
Gangloff et al.
,15
18
The azeotropically dried F-ion was reconstituted with a
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 269–283