Creatine kinase reactivation by thiol compounds

97

Clinwa Chimica A cta, 58 (1975) 97--99 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

CCA6566 CREATINE KINASE REACTIVATION BYTHIOLCOMPOUNDS

D.S. MIYADA, E.C. DINOVO* and R.M. NAKAMURA Department of Pathology, Orange County Medical Center, Orange, Calif. 92668 and The University of California, lrvine, Calif. 92664 (U.S.A.)

(Received August 13, 1974)

Introduction It is now c o m m o n l y accepted that thiol activated creatine kinase (CK) assay systems measure CK activity more accurately and more reproducibly than non-activated systems. However, some differences have arisen in the literature in regards to the nature and course of CK activation. Som e thiol c o m p o u n d s have been reported to yield higher e n z y m e activities than others. Dalai et al. [1] reported that m e r c a p t o e t h a n o l at 6.5 mM is suboptimal in the Siegel and Cohen assay [ 2 ] ; whereas, dithiothreitol (DTT) at 4 mM yields m axi m um activity. Warren has shown that DTT and m e r c a p t o e t h a n o l produce significantly greater CK activities than cysteine, d i t h i o e r y t h r i t o l (DTE), glutathione, or mercaptoacetate [3]. Bishop et al. [4] and Kar and Pearson [5] r e p o r t e d t h a t CK was equally activated i nde pe nde nt of the thiol activator. We report here our findings on the relative effectiveness o f m e r c a p t o e t h a n o l , cysteine, glutathione, and DTT in the reactivation of serum CK using the Oliver--Rosalki m e t h o d [6,7] and some characteristics of the reaction process.

Materials and Methods Human sera elevated in CK were pooled and served as the source for CK. Oxygen was bubbled through the pooled serum for 30--45 rain to inactivate CK. The inactivation for all practical purposes was complete. The thiol compounds, m e r c a p t o e t h a n o l , glutathione, cysteine, and dithiothreitol each at a final concentration 0.5, 1.0, 5.0 and 10.0 mM, were used to study the reactivation of CK. The Boehringer-Mannheim reagent system (No. 15926 TCAF) was used for this study because the activator is added separately in this system and allows, thus, the selective use o f different activators. Pooled serum, 100 pl, * Present address: D e p a r t m e n t o f P s y c h i a t r y and H u m a n B e h a v i o r , T h e U m v e r s l t y o f C a l i f o r n i a at I.rvlne, I r v i n e , Calif. 9 2 6 6 4 ( U . S . A . )

98 containing inactivated CK was added to 2.50 ml of substrate (containing triethanolamine buffer, 0.11 M, pH 7.0; D-glucose, 22 mM, magnesium acetate, 11 mM; adenosine diphosphate (ADP), 1.1 mM; adenosine monophosphate (AMP}, 11 mM; creatine phosphate, 38 mM) and 0.02 ml of a solution contain. ing the auxiliary enzymes hexokinase, 1 mg/ml and glucose-6-phosphate de. hydrogenase (GPD), 1 mg/ml. At zero time, 100 pl of thiol activator solution was added. The reactants were mixed and aspirated into the temperature. controlled cuvette. Absorbance measurements were obtained at 340 nm on the Gilford 300-N Spectrophotometer with Data Lister 4008. All reaction were carried out at 37 o including the pre-incubation of reagents. Results and Discussion A typical study showing absorbance changes with time for the reactivation of serum CK by glutathione is illustrated in Fig. 1. Similar patterns were obtained for DTT, cysteine, and mercaptoethanol. The lag times and the calculated enzyme activities are given in Table I. Several conclusions can be quickly delineated. Our results show that the four thiol compounds tested fully reactivate serum CK, and that CK activity increases with increasing activitor concentration until maximum activity is attained. Mercaptoethanol, cysteine, DTT, and g l u t a t h i o n e maximally activate CK at 1.0, 5.0, 10.0 and 10.0 mM, respectively. Therefore, in agreement with Bishop et al. [3] and Kar and Pearson [4], we find that all four thiol activators maximally reactivate CK, at differing concentrations, however. Although reaction linearity was observed for all activators and at all concentrations, the reaction rates with lower activator concentrations were only a fraction of the maximum rate; thus, at a glutathione concentration of 0.5 and 10.0 mM, CK activity was 337 and 759 U/l, respectively. This attainment of linearity without attaining m a x i m u m enzymatic activity was observed with all activator systems and we feel that it reflects either exhaustion of thiol activator or the attainment of an active--inactive e n z y m e equilibrium.

1500

5ram 1100

lOmM

ImM

~. I00: "nrne, m i n u t e s Fig. 1. T h e e f f e c t o f v a r y i n g g l u t a t h i o n e c o n c e n t r a t i o n s o n l a g t i m e u s i n g t h e O l i v e r - - R o s a l k i C K method.

99 TABLEI CK A C T I V I T Y A N D L A G T I M E V E R S U S T H I O L C O N C E N T R A T I O N

Thioltested

DTT Glutathione Cystein e

Thiol concentration (mmolfliter) 0.5

1.0

5.0

i0.0

Lag t i m e ( m i n )

590 11

717 8

721 3

768 1

Activity Lag t i m e ( r a i n )

337 14

616 12

713 5

759 3

Activity

438 7

675 6

781 3

818 1--2

548 13

793 9

785 4

784 2--3

Activity (U/liter)

Lag t i m e ( r a i n ) Mercaptoethanol

Activity

Lag t i m e ( r a m )

Lag phase has been defined as the time interval (lag time) for an enzyme reaction to attain linearity. It includes, therefore, the time necessary to activate the enzyme as well as the time to attain steady levels of intermediates when using a coupled enzyme system such as the Oliver--Rosalki m e t h o d for CK determination. Our results (Table I) show that lag time using the Oliver-Rosalki method for CK measurements is dependent upon the specific activator as well as its concentration. Lag time decreases with increasing concentrations of thiol activators; however, note that it is never completely eliminated and at an activator concentration of 10 mM, a lag time of one to three minutes is still needed to reach Iinearity. In agreement with an earlier report by Hess et al. [8], we also found lag time to vary with enzyme activity; decreasing with increasing e n z y m e activity. In view of the apparent dependence of lag time on the activator, activator concentration, and enzyme concentration, we feel the data to be of some significance in favoring the use of those instruments with positive checks on reaction linearity in preference to those using pre-set time intervals, all else being equal. References 1 F.R. Dalai, J. Cilley, J r a n d S. W i n s t e n , Clln. C h e m . , 1 8 ( 1 9 7 2 ) 3 3 0 2 A.L. Siegel a n d P.S. C o h e n , A u t o m a t i o n Ln A n a l y t i c a l C h e m i s t r y , T e c h n i c o n S y m p o s i a . M e d i a d . W h i t e Plains, N e w Y o r k , 1 9 6 7 , p p . 4 7 4 - - 4 7 6 3 W.A. W a r r e n , Clln. C h e m . 18 ( 1 9 7 2 ) 4 7 3 4 C. B i s h o p , T.M. C h u a n d Z . K . S h i h a b i , Clin. C h e m . , 17 ( 1 9 7 1 ) 5 4 8 5 N.C. K a r a n d C.M. P e a r s o n , P r o c . S o c . E x p . Biol. M e d . , 18 ( 1 9 6 5 ) 6 6 2 6 I.T. Oliver, B i o c h e m . J., 61 ( 1 9 5 5 ) 1 1 6 7 S.B. R o s a l k i , J. L a b . Clin. M e d . , 6 9 ( 1 9 6 7 ) 6 9 6 8 J.W. Hess, R . P . M c D o n a l d , G . J . W . N a t h o a n d K . J . M u r d o e k , Clin. C h e m . , 13 ( 1 9 6 7 ) 9 9 4