Avidin assay: A new procedure suitable for tissue fractions

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33 o19

Avidin assay: A new procedure suitable for tissue fractions Avidin, the biotin-binding protein of egg white, has been assayed until recently by the microbiological procedure of EAKIN, SNELL AND WILLIAMSz which is extraordinarily sensitive, but tedious and imprecise. As part of a series of studies of the chemical structure of avidin, GREEN described assay procedures based upon spectral shifts due to biotin 2 and dye 3 binding to avidin. GREEN4 and VC'EIAND WRIGHT5 have developed methods of greater sensitivity using radioactive biotin but they are either imprecise or tedious. Because of our interest in induction of avidin synthesis in chick oviducts, we have developed a simple, sensitive and precise assay system for avidin which is applicable to tissue homogenates. Chemicals were reagent grade. Distilled water from a central supply was further purified b y passage through an ion-exchange column. Bentonite was used as obtained. NCS Solubilizer was obtained from the Nuclear Chicago Corporation. Cellulose nitrate filters, 0.45 ~ pore size, were obtained from the Millipore Corporation. Purified avidin, prepared by the method of MELAMED AND GREEN6 was obtained from the Worthington Biochemical Corporation. By dye-binding assay 3 it had an activity of IO units/mg when freshly prepared and was used as a standard in all assays, v[carboxyl-14CJBiotinwas a gift of the Hoffmann LaRoche Company. A specific activity of 50 ~C/mg was determined by specific binding to avidin of known potency. A standard curve consisting of three or more concentrations of avidin was constructed for each assay. Standards and samples assayed were in duplicate or triplicate. Reagents and avidin samples were diluted in 0.2 M ammonium carbonate. The reaction mixture consisted of D-[~4Clbiotin plus standard or unknown in a final vol. of 0.6 ml. After IO min at 23 °, I ml of ammonium carbonate containing IO mg of bentonite was added. After 5 min, the suspension was transferred to a millipore filter under suction and rinsed once. Unbound biotin passed through the filter. The filter containing the bentonite-avidin-E~4Clbiotin complex was dissolved in a counting vial in IO ml of BRAY'S solution 7 leaving a bentonite sediment. Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer, Model 4000, at an efficiency of 85 % without quenching. Avidin was assayed in oviduct magnum homogenates obtained from estrogen and progesterone pretreated immature chicks. At the time of sacrifice, the magnum portion of the oviduct was homogenized in 6 to IO times its weight of buffer consisting of 0.07 M KC1, 0.07 M NaC1, o.004 M MgC12, 0.02 M sodium phosphate (pH 7.1) using a motor-driven teflon pestle. After preliminary centrifugation at 6000 × g for 15 min, samples underwent ultracentrifugation at 94000 × g for 9 ° rain and the supernatant used for avidin assay. In order to determine whether tissue biotin-binding activity was due to avidin, aliquots were reacted with E~4C~biotin and subjected to electrophoresis. Acrylamide-gel electrophoresis was performed by the method of REISFELD, LEWIS AND WILLIAMS8. Gels were sliced using the CHRAMBACH device 9. Slices were incubated overnight at 23 ° in I ml of NCS solubilizer which resulted in partial hydrolysis of the gel and complete release of [~*Cjbiotin counts. Radioactivity was counted after the addition of 5 ml of toluene phosphor. Biochim. Biophys. Acta, 14o (1967) I74-I76

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A typical standard dose response curve is illustrated in Fig. I. The index of precision, ,t was O.lO5. The 95 % confidence limits of an estimate of avidin concen40C

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A v i d i n (pg)

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0 I1 Or2 013 0~4 Tissue homogenote (ml)

Fig. I. Avidin assay s t a n d a r d curve. Dilutions of an avidin s t a n d a r d were assayed in triplicate. The curve was obtained from a regression analysis of the d a t a 1°. The index of precision ~ = s/b was O.lO 5 . Fig. 2. Avidin assay of tissue homogenates. Aliquots of oviduct h o m o g e n a t e s u p e r n a t a n t s p r e p a r e d as described in the t e x t were assayed. E a c h p o i n t represents the m e a n of duplicates which differed by less t h a n 2o c o u n t s / m i n in all cases.

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Slice number Fig. 3. Electrophoretic mobility of biotin-binding activity of oviduct s u p e r n a t a n t fractions. Radioactivity was m e a s u r e d in slices obtained from acrylamide-gel electrophoresis of an avidin s t a n d a r d and 4 o v i d u c t s u p e r n a t a n t s p r e p a r e d as described. Slice n u m b e r one was at the origin of the l o w e r gel. The biotin-binding activity which m i g r a t e d more rapidly t h a n the bulk of s u p e r n a t a n t p r o t e i n as determined b y staining of duplicates w i t h amidoblack was in a single peak in the same location in the tissue fractions (right) and s t a n d a r d (left).

Biochim. Biophys. Acta, 14o (1967) 174-176

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tration based on the mean of duplicates was ~= O.Ol5 t~g. The assay procedure, therefore, was much more precise than most biological assays. Fig. 2 shows that in tissue fractions [14C]biotin binding was proportional to volume. Since a standard curve using avidin assayed b y an independent method s was used in each assay, we were measuring avidin if, and only if the biotin-binding material in oviduct was chemically similar to avidin. Fig. 3 illustrates patterns of radioactivity in gel slices from electrophoresis of oviduct supernatants. The mobility of the biotin-binding activity was similar to that of authentic avidin. Furthermore, in Table I comparison of the radioactivity bound b y electrophoresis and by standard assay shows that all the oviduct biotin-binding activity had the mobility of avidin so that there could be no other binding protein in the supernatant. Thus, using a simple, rapid assay procedure, avidin can be measured precisely in concentrations found in the stimulated chick oviduct. TABLE I [14C]BIOTIN BINDING AND AVIDIN CONTENT OF TISSUE FRACTION'S OBTAINED BOTH BY STANDARD ASSAY AND BY ACRYLAMIDE-GEL ELECTROPHORESIS E q u a l a l i q u o t s (0.2 ml) of tissue s u p e r n a t a n t s were a s s a y e d a n d were s u b j e c t e d to a c r y l a m i d e - g e l e l e c t r o p h o r e s i s a f t e r r e a c t i n g w i t h an excess of !14C]biotin. Slices of t h e gel were c o u n t e d as described. The a m o u n t of [14C]biotin b o u n d b y b o t h m e t h o d s c o r r e c t e d to d i s i n t . / m i n is given. U n b o u n d b i o t i n did not e n t e r the gel.

Sample

Standard assay (disint./min)

Gel electrophoresis (disint./min)

A vidin content (lzg)

I 2 3 4

1192 1212 2o5 257

1182 144o 181 291

o.54 o.56 o.o6 o.o8

The outstanding and devoted technical assistance of Mrs. PATRICIA MIDDLETON is gratefully acknowledged.

Endocrinology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) I 2 3 4 5 6 7 8 9 io

STANLEY G.

KORENMAN*

B E R T W . 0'IV[ALLEY

R. E. EAKIN, E. I~. SNELL AND R. J. WILLIAMS,J. Biol. Chem., 14o (1941) 535. N. M. GREEN, Biochem. J., 89 (1963) 599. N. M. GREEN, Bioehem. J., 94 (1965) 23 C. N. M. GREEN, Biochem. J., 89 (1963) 585 . R. WEI AND L. D. ~VVRIGHT,Proc. Soc. Exptl. Biol. Med., 117 (1963) 17. M. D. MELAMED AND N. M. GREEN, Biochem. J., 89 (1964) 591. G. A. BRAY, Anal. Biochem., I (196o) 279. R. A. ]~EISFELD, U. J. L E w i s AND D. E. WILLIAMS, Nature, 195 (1962) 281. A. CHRAMBACH,Anal. Biochem., 15 (1966) 544. W. J. D i x o n AND F. J. MASSEY, Introduction to Statistical Analysis, Mc G ra w -H i l l , N e w Y o r k , N.Y., 1957, p. 191, folios.

Received February 8th, 1967 * P r e s e n t a d d r e s s : D e p a r t m e n t of Medicine, U.C.L.A. School of Medicine, H a r b o r H o s p i t a l C a m p u s , Torrance, Calif., 90509, U.S.A.

Biochim. Biophys. Aeta, 14o (1967) 174-176