562 Biochimica et Biophvsica Acta 924 (1987) 562- 563 Elsevier

BBA 20205 BBA Report

Occurrence of vanadium ion in serum albumins

Hiromu Sakurai a, Mikio Nishida a, Mutsuo Koyama b and Jitsuya Takada b Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima and b Research Reactor Institute,

Kyoto University, Osaka (Japan)

(Received 24 November 1986) (Revised manuscript received 6 April 1987)

Key words: Bovine serum albumin; Vanadium content; Neutron activation analysis; Vanadate ion; Vanadyl ion

Vanadium was shown to be a contaminant in Sigma-grade serum albumins, fraction V. The levels of vanadium detected by neutron activation analysis were: bovine 16.7, ovine 10.0, rabbit 9.1, porcine 3.9, and human 0.19 pg/g protein. According to the ESR spectra, the vanadate form (-I-5 oxidation state) was strongly suggested as a chemical form present in albumins. Dialysis against a chelating agent was quite effective for removal of the metal ion.

Since vanadium ion is known to be an essential trace element in many animals [1-3] and is a potent inhibitor of (Na + + K +)-ATPase at physio- logical concentrations [4], much interest has been focused on the physiology and biochemistry of this element. However, because of the difficulty in obtaining accurate measurements of vanadium ion levels and states in biological materials, reliable information on the concentrations of vanadium ion in animals is limited.

Recently, vanadium concentrations have been reported in human sera [5-7] and marine biologi- cal samples [8] by neutron activation analysis, which is the most reliable method currently used for the determination of vanadium.

During chemical investigations of vanadium ion, we found that vanadium ion is a contaminant in commercially available bovine serum albumins.

Vanadium contents in protein samples were determined by a neutron activation analysis

Correspondence: H. Sakurai, Faculty of Pharmaceutical Scien- ces, University of Tokushima, Sho-machi 1, Takushima 770, Japan.

method. The gamma-ray spectrogram of Sigma- grade serum albumin showed that 52V (half-life, 3.75 min) gave rise to an easily detectable photon peak at 1434.0 keV, being separated from the peaks of 24Na and 38C1. The 1434.0 keV peak was used to calculate the vanadium concentration in albumins.

The concentration of vanadium in commer- cially available serum albumins, purified albumins after dialysis, and bovine trypsins are summarized in Table I. In Sigma-grade bovine serum albumin, fraction V, 16.7 #g vanadium/g protein were de- tected, whereas in the albumin after dialysis, the amounts were decreased to 11-14% of this amount. The concentration of vanadium in Boehringer- grade bovine serum albumin was estimated as 0.19 /~g/g protein.

The level of vanadium in Sigma-grade serum albumins from various sources were in the follow- ing order: bovine > ovine > rabbit > porcine > human. Vanadium was also detected in Sigma- grade trypsins, particularly in the type III-s sam- ple (5.2/~g/g protein).

The ESR spectrum at 77 K of Sigma-grade bovine serum albumin dissolved in 0.1 M Tris-HC1

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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TABLE I

VANADIUM CONTENTS IN ALBUMINS AND TRYP- SINS

A 1 mM solution of bovine serum albumin was dialysed at 4°C against 0.01 M o-plienanthroline or EDTA, pH 6.0, for 20 h, followed by several changes of distilled deionized water for 3 days. It was then lyophilized. BSA represents bovine serum albumin.

Protein Vanadium (#g/g protein)

BSA, fraction V (Sigma, A4503) 16.69 + 0.88 (n = 7)

BSA dialysed against: o-phenanthroline 1.98 ( n = 2) EDTA 2.35 (n = 2)

BSA, fraction V (Boehringer, 735078) 0.19 (n = 2)

BSA dialysed against: o-phenanthroline 0.15 (n = 2) EDTA 0.10 (n = 2)

Albumins (Sigma) human (A1653) 0.19 (n = 2) porcine (A2764) 3.87 (n = 2) rabbit (A0639) 9.10 (n = 2) ovine (A3264) 10.00 (n = 2)

Bovine trypsin (Sigma) type III-s (T2395) 5.23 (n = 2) type I (T8003) 0.96 (n = 2)

buffer (pH 7.5) showed the absence of vanadyl ion. However, when a reducing agent such as ascorbic acid or cysteine was added to the al- bumin solution, a relatively intense signal com- posed of eight lines appeared [9]. Thus it was confirmed that the vanadium ion detected in bovine serum albumin is in the vanadate (+5 oxidation state) form.

The binding profile of vanadium ion to bovine serum albumin is not certain, but approx. 85% of the metal in the protein is easily removed by dialysis against o-phenanthroline or EDTA. 15% of the total vanadium may, therefore, be assumed to bind more strongly to albumin.

The number of binding sites of bovine serum albumin and trypsin to vanadium ion have been reported to be 40 and 14, respectively [10]. On the

basis of this result, bovine serum albumin (M r 69000) and trypsin (M r 24000) bind vanadium ion at a maximal level of 28.7 and 28.8 /xg/g protein, respectively, indicating that the binding sites are not fully saturated by the vanadium ion.

Serum albumins have been used in a variety of ways in many fields; e.g., as standard material for protein determination, fatty acid carrier for cell cultures and as protecting material for macro- phage separation. Vanadium ion above trace ele- ment levels in serum albumins cannot be ne- glected in use, because it may influence any bio- chemical reactions. The present results point out that commercially available serum albumins should be at least dialysed before use.

Acknowledgments

This work has been carried out under the Visit- ing Researcher's Program of the Research Reactor Institute of Kyoto University.

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