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Chapter 8 Enzymes
B6 3
Chapter 8 ENZYMES 8.1 I I ~ T R O D U C T I O N
Enzymes are p r o t e i n s and t h e r e f o r e the methods and problems described and discussed i n Chapter 7 are v a l i d a l s o f o r enzymes. However, t h e r e a r e some special features t h a t must be emphasized when the separation o f enzymes i s considered. I n general, enzymes are o f t e n very s e n s i t i v e t o denaturation, u s u a l l y r e s u l t i n g i n a t o t a l o r p a r t i a l l o s s o f c a t a l y t i c a c t i v i t y . The y i e l d o f an enzyme f r a c t i o n a t i o n must be expressed n o t o n l y i n terms o f t h e p r o t e i n recovery, b u t a l s o i n terms o f the recovery o f enzymic a c t i v i t y . The increase i n the r a t i o of the a c t i v i t y t o the p r o t e i n content i s the usual measure o f the degree o f p u r i f i c a t i o n i n the process o f enzyme i s o l a t i o n . The a c t i v i t y / p r o t e i n r a t i o i s a1 so an important constant c h a r a c t e r i z i n g the pure and homogeneous enzyme preparation. The r i s k o f denaturation o f enzymes a r i s e s n o t o n l y w i t h elevated temperature. F r a c t i o n a t i o n a t temperatures several degrees above zero i s u s u a l l y used n o t only t o e l i m i n a t e the p o s s i b i l i t y o f thermal denaturation o f enzymes, b u t a l s o because many raw m a t e r i a l s o f b i o l o g i c a l o r i g i n contain c e r t a i n amounts o f various proteinases, which can p a r t l y decompose the enzymes t o be i s o l a t e d , e s p e c i a l l y a t the usual ambient temperatures. Unknown enzyme preparations should c e r t a i n l y n o t be exposed t o extreme c o n d i t i o n s o f pH, which very e a s i l y change t h e i r t e r t i a r y structure. Organic solvents represent another t h r e a t t o the a c t i v i t y o f some enzymes e s p e c i a l l y a t elevated temperatures. This must be considered when reversed-phase high-performance 1 i q u i d chromatography (RP-HPLC) experiments a r e planned. The same applies t o detergents: sodium dodecylsulphate s o l u t i o n i s useful f o r t h e determination o f molecular weights of enzymes using t h e s i z e exclusion chromatography (SEC) mode, b u t t h e a c t i v i t y i s destroyed. Some enzymes form special quaternary s t r u c t u r e s and the detergents d i s s o c i a t e t h i s s t r u c t u r e i n t o i n d i vidual subunits. Other surface-active compounds a l s o endanger enzymes. Many enzymes cannot t o l e r a t e c o n t a c t w i t h ions o f heavy metals, and t h i s must be borne i n mind when b u f f e r s a r e selected f o r t h e e l u t i o n o f enzymes from s t a i n less-steel chromatographic columns: some b u f f e r s a t t a c k the s t a i n l e s s s t e e l (e.g.,
a c i d i c s o l u t i o n s o f ammonium formate) and t r a c e amounts o f heavy metal
B64 i o n s can d e s t r o y t h e enzyme a c t i v i t y i n some i n s t a n c e s ; g l a s s o r o t h e r nonc o r r o s i v e columns and pumps a r e t o be p r e f e r r e d . Oxygen i n t h e a i r ( a l s o d i s s o l v e d i n t h e m o b i l e phase) can d e a c t i v a t e some s e n s i t i v e enzymes. I n p a r t i c u l a r , t h e enzymes o f anaerobic microbes and many o t h e r enzymes o f anaerobic metabolism may be s e n s i t i v e t o oxygen t o such an e x t e n t t h a t t h e a d d i t i o n o f r e d u c i n g agents t o t h e m o b i l e phase i s n o t s u f f i c i e n t t o p r o t e c t t h e a c t i v i t y and t h e s e p a r a t i o n must be performed i n a s p e c i a l anaero b i c l a b o r a t o r y . S t r o n g r e d u c i n g agents w i l l a l s o d e s t r o y t h e a c t i v i t y o f some enzymes. S u r f a c e d e n a t u r a t i o n o f enzymes i n c o n t a c t w i t h u n s u i t a b l e chromatographic sorbents t h a t a l l o w s t r o n g hydrophobic i n t e r a c t i o n s has o f t e n been observed; h y d r o p h i l i c adsorbents a r e p r e f e r a b l y used. F o r t h i s reason, t h e n e w l y developed h y b r i d h y d r o p h i 1 i c / h y d r o p h o b i c pac k i n g s f o r hydrophobic i n t e r a c t i o n c hromatography (HIC) o f enzymes a r e v e r y i m p o r t a n t ( c f . ,
S e c t i o n s 3.6 and 4.4.3).
The t o t a l l o s s o f enzymic a c t i v i t y due t o t h e d e n a t u r a t i o n o f n a t i v e enzymes i s n o t t h e o n l y r i s k when enzymes a r e separated. What may be n e g l e c t e d a r e s l i g h t changes i n c o n f o r m a t i o n , o r 1 i m i t e d p r o t e o l y s i s , which s l i g h t l y m o d i f y enzymes so t h a t t h e y d i f f e r i n t h e i r chromatographic r e t e n t i o n f r o m t h e o r i g i n a l n a t i v e form, b u t s t i l l r e t a i n t h e i r a c t i v i t y . A r t i f i c i a l enzyme polymorphism i s c r e a t e d i n t h i s way and i n s t e a d o f n a t i v e enzymes t h e a c t i v e a r t i f a c t s may be i s o l a t e d . " A r t i f i c i a l isoenzymes" a r e formed t h a t can h a r d l y be d i s t i n g u i s h e d f r o m n a t u r a l isoenzymes a t t h e b e g i n n i n g o f t h e s t u d y because m u l t i p l e forms o f enzymes v e r y o f t e n o c c u r i n n a t u r e . S p e c i a l r e q u i r e m e n t s f o r t h e low-speed chromatography o f enzymes were d i s cussed f r o m t h i s p o i n t o f view, e.g.,
by Mikes (1975).
Enzymes a r e b i o c h e m i c a l c a t a l y s t s and t h e i r a c t i v i t i e s have been measured by many workers i n v a r i o u s ways. I t was e x t r e m e l y d i f f i c u l t t o compare r e s u l t s r e p o r t e d on t h e same enzyme. Hence t h e F i f t h I n t e r n a t i o n a l Congress o f Biochemi s t r y adopted t h e recommendations o f t h e Commissions on Enzymes o f I U P A C ( I n t e r n a t i o n a l Union o f Pure and A p p l i e d C h e m i s t r y ) and IUB ( I n t e r n a t i o n a l Union o f B i o c h e m i s t r y ) (1965, 1972) f o r t h e d e f i n i t i o n o f an enzyme u n i t . The recommended terms w i l l be b r i e f l y p r e s e n t e d h e r e a c c o r d i n g t o G u i l b a u l t (19761, as follows. One unit l U ) of any enzyme i s t h a t amount which w i l l c a t a l y s e t h e t r a n s f o r m a t i o n o f 1 vmol o f s u b s t r a t e p e r m i n u t e ; where more t h a n one bond o f each s u b s t r a t e i s a t t a c h e d , t h e t r a n s f o r m a t i o n o f 1 uequiv. of t h e group concerned p e r m i n u t e i s considered, under d e f i n e d c o n d i t i o n s . Recommended parameters: temperature, 25OC; pH, o p t i m a l ; r e a c t i o n r a t e , i n i t i a l ; and k i n e t i c s w i t h r e s p e c t t o t h e s u b s t r a t e , zero o r d e r .
865
Concentration o f an enzyme i n s o l u t i o n : u n i t s p e r m i l l i l i t r e (U/ml) o r p e r l i t r e (U/l).
S p e c i f i c a c t i v i t y o f an enzyme: u n i t s o f enzyme p e r m i l l i g r a m o f p r o t e i n (U/mg p r o t e i n ) .
Molecular a c t i v i t y : u n i t s p e r micromole o f enzyme, i.e.,
t h e number o f mole-
c u l e s o f t h e s u b s t r a t e t r a n s f o r m e d p e r m i n u t e p e r m o l e c u l e o f t h e enzyme. 107 m e k a t a l r e f e r s t o t h e c o n v e r s i o n o f 1 mol o f s u b s t r a t e p e r second = 6 7 pmol o f s u b s t r a t e p e r m i n u t e = 6 10 u n i t s (one u n i t = 1 U = 16.67 n k a t a l ) . PRINCIPLES OF SPECIFIC DETECTION METHODS FOR ENZYMES AND ISOENZYMES
8.2.
One o f t h e s i m p l e s t approaches i s t o measure t h e absorbance i n t h e long-wave
UV r e g i o n ( P r u s i k , 1975), which i s used f o r t h e s e n s i t i v e d e t e c t i o n o f some enzymes w i t h f i r m l y bound p r o s t h e t i c groups. However, when d e a l i n g w i t h t h e f r a c t i o n a t i o n o f c r u d e m i x t u r e s o f p r o t e i n s , s p e c i f i c enzymes must b e quant i t a t e d by enzyme assay o f f r a c t i o n s r a t h e r t h a n by s p e c t r o p h o t o m e t r i c m o n i t o r i n g o f t h e e f f l u e n t . The chromatographic column e f f l u e n t can be analysed u s i n g an o f f - l i n e o r an o n - l i n e procedure. F o r t h e o f f - l i n e procedures a l i q u o t s o f t h e f r a c t i o n s a r e t a k e n m a n u a l l y o r by some a u t o m a t i c equipment (see, e.g.,
a short
r e v i e w i n an essay by Mikes, 1975). Raschbaum and Everse (1978) a l s o d e s c r i b e d an apparatus f o r t h e a u t o m a t i c d e t e c t i o n o f enzyme a c t i v i t y i n column chromatog r a p h i c e f f l u e n t s , which c o n s i s t s o f a s i m p l e s t o p p e d - f l o w i n s t r u m e n t , equipped w i t h a m u l t i - s h u t o f f v a l v e t o d i r e c t t h e r e a g e n t and e l u e n t f l o w s : assays o f t h e e l u a t e can be c a r r i e d o u t a t p r e - s e t i n t e r v a l s . The chemical p r i n c i p l e s and a n a l y t i c a l procedures f o r t h e q u a n t i t a t i o n o f many i m p o r t a n t enzymes were d e s c r i b e d , e.g.,
i n t h e handbook by G u i l b a u l t (1976).
From t h e p o i n t o f v i e w o f t h e p r e s e n t s t a t e o f e v o l u t i o n o f t h e l i q u i d c h r o matography o f enzymes, o f f - l i n e d e t e c t i o n procedures seem t o be u s e f u l o n l y f o r p i o n e e r works i n a t t e m p t s t o i s o l a t e an unknown enzyme, o r w i t h some s i n g l e a c t i o n p r e p a r a t i v e experiments i n o r d e r t o i s o l a t e a known r e q u i r e d enzyme, and never f o r r o u t i n e l y r e p e a t e d a n a l y t i c a l o r p r e p a r a t i v e procedures. When HPLC s e p a r a t i o n s o f p r o t e i n s i n 10 min became p o s s i b l e , time-consuming o f f - l i n e enzymic assays o f t h e components were t h e l i m i t i n g s t e p . T h e r e f o r e , r a p i d o n - l i n e d e t e c t i o n methods f o r enzymes and isoenzymes were sought. The i n s t r u m e n t a t i o n o f d e t e c t o r s developed f o r t h i s purpose was d e a l t w i t h i n d e t a i l i n S e c t i o n 5.2.3.
I n t h e f o l l o w i n g sections the biochemical p r i n c i p l e s o f t h e s p e c i f i c
d e t e c t i o n methods w i l l be e x p l a i n e d . T h i s approach was e s p e c i a l l y i m p o r t a n t i n c l i n i c a l a p p l i c a t i o n s f o r t h e i d e n t i f i c a t i o n o f isoenzymes. M e i s t e r (1950) and N e i l a n d s (1952) were t h e f i r s t t o r e c o g n i z e m u l t i p l e forms o f t h e enzyme l a c t a t e dehydrogenase ( L D ) . Vessel
666 and Bearn (1957) opened a new dimension i n c l i n i c a l diagnosis by t h e i r f i n d i n g t h a t the serum p r o f i l e of LD isoenzymes changes d r a m a t i c a l l y w i t h myocardial i n f a r c t i o n o r acute myelogenous leukaemia. Cohen e t a l . (1964) described i n d e t a i l serum LD patterns i n cardiovascular and other diseases, w i t h p a r t i c u l a r reference t o acute myocardial i n f a r c t i o n . The diagnostic importance o f isoenzymes has been evaluated i n many papers (e.g.,
Galen e t al., 1975). Usually, isoenzymes
were separated from each other using mainly e l e c t r o p h o r e t i c , ion-exchange, immunochemical and various s o r p t i o n techniques. Morin (1977) evaluated the methods c u r r e n t a t t h a t time f o r c r e a t i n e kinase isoenzyme f r a c t i o n a t i o n . Kudirka e t a l . (1975) were the f i r s t t o apply modern r a p i d HPLC separation t o c r e a t i n e kinase isoenzymes, b u t the o n - l i n e detector was missing. However, the authors discussed i t s importance.
A new p o s s i b i l i t y f o r the development o f serum isoenzyme p r o f i l i n g was opened w i t h the i n t r o d u c t i o n o f HPLC methods i n t o p r o t e i n chemistry and biochemistry. The speed o f the separation i n i t i a t e d a search f o r an equivalent r a p i d o n - l i n e detection method. The d i a g n o s t i c a l l y important isoenzymes were separated from each other on HPLC columns, b u t were n o t separated from various a d d i t i o n a l serum proteins , so t h a t detection methods were necessary t h a t would be able t o "see" the isoenzymes b u t n o t the other proteins covering t h e i r peaks. The biochemical p r i n c i p l e s of such techniques a r e described i n the f o l l o w i n g sections. A l k a l i n e phosphatase
8.2.1
The s p e c i f i c detection o f a l k a l i n e phosphatase (AP) represents t h e f i r s t simple example on which on-line post-column detection can be explained (Chang e t al.
,
1976; Schlabach e t a l .
, 1977;
Schlabach and Regnier, 1978). The a n a l y t -
i c a l column e f f l u e n t i s mixed w i t h the substrate p-nitrophenyl phosphate (NPP) i n the equipment i l l u s t r a t e d i n Fig. 5.29, and the mixture i s heated (4OoC) i n the packed column reactor i n which t h e product p-nitrophenol (NP) i s formed according t o the equation o2NC6H4oPo3Na2 NPP
AP
H20
02NC6H40H + HOP03Na2 NP
The product (NP) can be r e a d i l y detected i n a l k a l i n e s o l u t i o n by measurement o f the absorbance a t 400 o r 410 nm, and the presence o f proteins (Amax 280-285 nm) has no effect. An example o f such an analysis i s i l l u s t r a t e d i n Fig. 5.30. I n a d d i t i o n t o the s e l e c t i v i t y , t h i s detection p r i n c i p l e i s more than 20 times more s e n s i t i v e than conventional absorbance monitoring o f p r o t e i n s a t 280 nm.
If a wider r e a c t o r column (250 x 8 mm 1.0.) was used and the temperature increased t o 6OoC, t h e detection l i m i t was 25 ng/ml o f a l k a l i n e phosphatase (Regnier e t a l . , 1977). 8.2.2
m p s i n and chymotrypsin
Another simple reaction s u i t a b l e f o r on-line post-column detection i s the spectrophotometric determination of trypsin (TRY), studied f o r t h i s purpose by Schlabach e t a l . (1977). Bender e t a l . (1966) described a number of stoichiometr i c reagents t h a t were developed f o r t h e t i t r a t i o n o f a c t i v e s i t e of enzymes and made i t possible t o determine the concentration o f an enzyme in solution with an end-point reaction. A very e f f e c t i v e a c t i v e s i t e t i t r a n t f o r TRY i s n i t r o phenyl guanidinoboenzoate (NPGB) (Chase and Show, 1967), which r e a c t s with TRY according t o t h e equation
H2N\//
NHbHCI
+
TRY-
The p-nitrophenol ( N P ) product can be monitored by spectrophotometry a t 400 nm. NPGB r e a c t s only with a c t i v e TRY and i s i n s e n s i t i v e t o o t h e r enzymes and t o i n a c t i v e TRY. Schlabach e t a l . (1977) demonstrated t h e l i n e a r i t y of t h e response and the s u i t a b i l i t y of the method f o r q u a n t i t a t i n g TRY in a flow-through r e a c t o r . The addition of NaCl a t a 0.5 M concentration t o the measured solution was recommended in order t o suppress t h e adsorption o f TRY on the equipment surfaces.
868 Another post-column o n - l i n e detection method f o r TRY was pub1 ished by Gooding e t a l . (1984). The e x i t from the a n a l y t i c a l HI-HPLC column was connected, using a union tee, w i t h the substrate pump and a packed column r e a c t o r containing SynChropak PCR (a m i c r o p a r t i c u l a t e non-porous packing). The e f f l u e n t from t h e reactor was monitored a t 410 nm. The substrate f o r TRY pumped t o the union t e e was benzoyl-D,L-arginine
p - n i t r o a n i l i d e (BANA) , dissolved i n 0.1 M potassium
phosphate s o l u t i o n (pH 7 ) . BANA i s hydrolysed t o p - n i t r o a n i l i n e (NA) by TRY (Hoverbach e t al.,
1960) according t o the equation
BANA
/NH C=NH
\
NH2
This i s a normal t r y p t i c c a t a l y s i s and n o t a s t o i c h i o m e t r i c r e a c t i o n as was the case i n the first-mentioned equation. A s i m i l a r method was used by Gooding and Schmuck (1983) f o r the post-column
on-1 i n e monitoring o f chymotrypsin (CHY).
Glutaryl-L-phenylalanine p - n i t r o -
a n i l i d e (GPNA) was used as a s p e c i f i c substrate, which i s hydrolysed by chymot r y p s i n t o p - n i t r o a n i l i n e (NA) (Remy e t al.,
1981) according t o the equation
B69
GPNA
The e f f l u e n t was monitored by t h e measurement o f the absorbance a t 410 nm. 8 . 2 . 3 Lactate dehydrogenase isoenzymes
The most i n t e n s i v e l y st udied isoenzyme f a m i l y are l a c t a t e dehydrogenases, owing t o t h e i r importance f o r diagnost ic purposes. Lactate dehydrogenase (LD) i s a tetramer o f Mr = 150 000, cont aining two s t r u c t u r a l l y d i s t i n c t subunits (M from s k e l e t a l muscle and H from h e a r t muscle). Various combinations o f these subunits l e a d t o f i v e d i f f e r e n t isoenzymes o f the same a c t i v i t y , LDHl
-
LDH5
H4, H3M, H2M2, HM3 and M4, r e s p e c t i v e l y ) . A t h i r d subunit, C, was found i n t e s t i c u l a r t i s s u e and spermatozoa. C u s u a l l y occurs as tetramer C4, b u t some (i.e.,
h y b r i d i z e d C+H+M isoenzymes were a l s o found i n nature (Evrev, 1975). The o x i d a t i o n o f l a c t a t e t o pyruvate using the oxidized form o f nicotinamide adenine di nucl eot ide, NAD',
i s t he p r i n c i p a l r e a c t i o n used i n a l l instances f o r
the post-column det ect ion o f these isoenzymes: C H~C H (OH )C OOH + NAD+
LD
C H ~ C O C O O H + NADH + H+
(*3407 E457) The reduced c o f a c t o r , NADH, can be cont inuously monitored e i t h e r by measurement
of the absorbance a t 340 nm o r by fluorescence ( e x c i t a t i o n a t 457 nm). The fluorescence method has t he g r e a t advantage o f higher s e n s i t i v i t y . More enzymec a t a l yse d reacti ons can be coupled t o t h i s redox reagent (cf.,
Section 8.2.4).
Kudirka e t a l . (1976) used f o r t he d e t e c t i o n o f LD a f t e r anion-exchange HPLC separation the above r eact ion, r e a l i z e d w i t h a Technicon AutoAnalyzer I1 s i n g l e channel col ori met er , where t he absorbance a t 340 nm was measured. Chang e t a l . (1976) used th e p r i n c i p l e i l l u s t r a t e d i n Fig. 5.29 (i.e.,
the supply o f the
s u bstrate through a union tee i n t o t he l i n e leading t o a packed r e a c t i o n column). I n contrast, Schroeder e t a l . (1977) described the p r i n c i p l e o f single-stream
'1 I "i
B
133 U 0
I2
LD2
L,
+
O! 0
I I
I
I
I I
TIME, MIN
I I
I
I
I I
I 32
Fig. 8.1. (A ) Chromatogram of serum LD isoenzymes from a patient with myocardial infarct. The upper trace was recorded a t the downstream detector (detector 2). The lower trace shows t h e background absorbance, a s i t was observed a t the upstream detector (detector 1 ) ( c f . , Fig. 5.31). The total serum LD a c t i v i t y was 502 U/l. (B) Profile of serum LD isoenzymes resulting from the correction f o r background absorbance (cf., A ) . The percentages of the total area a r e a s follows: LD5, 2.5; LD4, 1.2; LD3', 6.5; LD3, 4.4; LD2, 28.4; LDI, 56.9%; the high values of LDI and LD2 a r e significant f o r the diagnosis of infarction. (Reprinted from Ful t o n e t a l . , 1979.)
B71 o r p a r a l l e l -stream r e a c t i o n detectors based on an open-tubular system (an empty r e a c t i o n c o i l 1. Both absorbance and fluorescence monitoring were used. The postcolumn on-line detection o f LD isoenzymes was f u r t h e r i n v e s t i g a t e d and developed by Schlabach and co-workers (1977, 1978, 1980a,b) , Schlabach and Regnier ( 1 9 7 8 ) , Denton e t a l . (1979) and Fulton e t a l . (1979). L i v e r o r muscle damage, myocardial damage and some other accidents and pathological cases can be q u i c k l y detected by LO chromatographic p r o f i l i n g o f a small sample o f serum. Fig. 8.1 shows the r e s u l t o f the a p p l i c a t i o n o f a computer-controlled dual-detector post-column r e a c t i o n system t o LD serum p r o f i l i n g using background subtraction. D e t a i l s o f t h e various d e t e c t o r systems were discussed i n Section 5.2.3. LD chromatographic p r o f i l i n g w i t h o n - l i n e post-column d e t e c t i o n methods was reviewed by Regnier e t a l . ( 1 9 7 7 ) , Regnier and Gooding (1981) and Vacik and Toren ( 1982). 8.2.4
Hexokinase isoenzymes and 3-phosphogZycerate kinase
The post-column o n - l i n e detection o f hexokinase isoenzymes i s an example o f coupled enzyme assays. The enzyme monitoring methods can be c l a s s i f i e d i n t o two groups: (a) d i r e c t detection, where t h e immediate enzymic product i s monitored (Sections 8.2.1
- 8.2.3
g i v e several examples); (b) coupled enzyme assays,
where an a d d i t i o n a l enzyme o r more a d d i t i o n a l enzymes are required t o convert the product o f the primary enzyme r e a c t i o n i n t o a more e a s i l y detectable form. Schlabach and Regnier (1978) described the f i r s t method and a l s o the a p p l i c a t i o n o f the second method t o the detection o f hexokinase (HK) isoenzymes. They a l s o evaluated the e f f i c i e n c y o f both f r e e and immobilized coupling enzyme(s1. The measured hexokinase catalyses t h e phosphorylation o f D-glucose t o D-gl ucose-6-phosphate (G-6-P) using adenosine-5-triphosphate
(ATP) , and
adenosine-5-diphosphate (ADP) i s released. The G-6-P formed i s then o x i d i z e d by nicotinamide adenine dinucl e o t i d e (oxidized form NAD')
t o g l uconolactone-6-
phosphate; t h i s r e a c t i o n i s catalysed by a coupling enzyme, glucose-6-phosphate dehydrogenase (G-6-PDH). The reduced coenzyme NADH can be determined by known methods ( c f .
, Section
8.2.3),
i.e.
, by
absorbance measurement a t 340 nm or,
b e t t e r , by fluorescence measurement a t 457 nm. The sequence o f reactions i s as f o l 1ows : HK
D-Glucose + ATP -D-G1ucose-6-phosphate 0-G1 ucose-6-phosphate + NAD'
G-6-PDH
+ ADP
NADH + D-G1 uconolactone-6-phosphate + H+
(A340)
The coupling enzyme G-6-PDH must be added together w i t h the primary substrate D-glucose and other reagents (ATP + NAD')
t o t h e s o l u t i o n pumped i n t o t h e reactor.
However, the coupling enzyme i s o n l y a c a t a l y s t and i s n o t consumed i n t h e react i o n . It can be saved when used i n the r e a c t o r i n an immobilized form. This i s the general p r i n c i p l e of how t o save the a u x i l i a r y enzymes, which a r e sometimes expensive, i f they have t o be added t o the substrate s o l u t i o n i n a r e l a t i v e l y l a r g e amount and then f l o w from t h e r e a c t o r t o waste. Hexokinase isoenzymes were detected i n r a t l i v e r and t e s t i c u l a r t i s s u e e x t r a c t s using immobilized G-6-PDH i n a column reactor. For t h e detection o f HK, Lowe e t a l . (1981) used i n t h e i r "universal assay medium" t h e oxidized form o f nicotinamide adenine d i n u c l e o t i d e phosphate (NADP') instead o f NAD'
i n the above coupling reaction, and t h e reduced form NADPH was
obtained as the r e a c t i o n product, which absorbed a t 340 nm. For the detection of 3-phosphoglycerate kinase (PGK) a c t i v i t y , the f o l l o w i n g sequence o f reactions was used, where the coupling enzyme glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) was applied: 3-Phosphoglycerate + ATP
PGK
Glycerate-I ,3-biphosphate + ADP
Glycerate-l,3-biphosphate + NADH + H+
G-3-PDH
Glyceraldehyde-3-phosphate
t
(A340) t
NAD' + Phosphate
The "universal" assay medium contained both NADP' ( i n the oxidized) and NADH ( i n the reduced) form. I t i s evident t h a t the presence o f hexokinase i n t h e column e f f l u e n t produces an increase i n absorbance a t 340 nm, whereas 3-phosphoglycerate kinase a c t i v i t y decreases the absorbance a t 340 nm. The problems connected w i t h t h i s approach were discussed. 8.2.5
Creatine kinase isoenzymes
Creatine kinase (CK) i s a diiner o f Mr = 86 000 and i s composed o f M (muscle) o f B ( b r a i n ) subunits. Sometimes t h i s enzyme was designated c r e a t i n e phosphokinase, CPK. Three isoenzymes, CK, ( o r CPK,) t o CK3 (CPK3), can be derived from subunit combinations, i.e.,
BB, MB and MM, r e s p e c t i v e l y . CK i n b r a i n c o n s i s t s
o f BB o r contains B subunit, whereas MM predominates i n s k e l e t a l muscle and MB i s t y p i c a l of h e a r t muscle. CK isoenzyme p r o f i l i n g i s very important f o r the precise diagnosis o f myocardial i n f a r c t i o n , i n which MB isoenzyme i s elevated, i n c o n t r a s t t o the s i t u a t i o n i n coronary i n s u f f i c i e n c y .
CK c a t a l y s e s t h e phosphoryl a t i o n o f c r e a t i n e u s i n g adenosine-5-triphosphate (ATP), and t h e r e f o r e t h e systematic name o f t h i s enzyme i s ATP:creatine phosphotransferase. However, t h i s r e a c t i o n tends t o go i n t h e o p p o s i t e d i r e c t i o n : CK 0 Phosphocreatine + ADP I Creatine + ATP; AG = -3 kcal/mol The l i b e r a t e d ATP can be determined i n d i f f e r e n t ways. A method u s i n g a b i o luminiscence r e a c t i o n w i t h f i r e f l y l u c i f e r i n ( B o s t i c k e t a l . t i o n e d i n Section 6.6.4.
1980) was men-
Other methods employing c o u p l i n g enzymes w i l l be
described here. ATP can r e a c t w i t h 0-glucose using c a t a l y s i s by hexokinase (HK) and t h e D-glucose-&phosphate formed can be determined by o x i d a t i o n w i t h nicotinamide adenine d i n u c l e o t i d e (NAD')
(Sections 8.2.3
and 8.2.4),
or with
nicotinamide adenine d i n u c l e o t i d e phosphate m A D ( P ) + I :
ATP + D-glucose
HK -+
D-glucose-6-phosphate + ADP
0-G1 ucose-6-phosphate + NAD( P)'
G-6-PDH
D-gluconolactone-6-phosphate +
+ NAD(P)H+ H+ Schlabach e t a l . (1977) and Schlabach and Regnier (1978) described t h i s procedure. The use o f e i t h e r NAD' o r NAD(P)+ depends on whether t h e G-6-PDH c o u p l i n g enzyme i s from yeast o r from t h e microbe Leuconostoc mesenteroides. The l a t t e r p r e f e r s NAD',
whereas t h e y e a s t enzyme i s s p e c i f i c f o r NAD(P)+. The authors a l s o
t e s t e d and discussed t h e a p p l i c a t i o n o f immobilized c o u p l i n g enzymes. Denton e t a l . (1978, 1979) described t h e o n - l i n e m o n i t o r i n g of CK isoenzymes by use o f an immobilized enzyme microreactor. Other c o n t r i b u t i o n s t o t h e development o f CK post-column d e t e c t i o n were published by Schlabach e t a l . (1978, 1980a).
Interferences appearing i n t h e f l u o r i m e t r i c a l l y measured p r o f i l e s o f CK i s o enzymes i n human serum, due t o serum albumin, l i p o p r o t e i n and prealbumin, were discussed by Schlabach e t a l . (1980b). E l k i n s (1977) described an a l t e r n a t i v e method f o r t h e determination o f CK isoenzyme a c t i v i t y by t h e a p p l i c a t i o n o f an anion-exchange column and a c e n t r i f u g a l analyser. The HPLC o n - l i n e m o n i t o r i n g o f CK isoenzymes was discussed and reviewed by Regnier e t a l . (1977) , Regnier and Gooding (1981) and Vacik and Toren (1982).
874
8.2.6
ArylsuZphatase isoenzymes
Bostick e t a l . (1978) described t h e anion-exchange separation o f two a r y l sulphatase isoenzymes, A and B y i n human u r i n e using continuous d e t e c t i o n based on p-nitrocatechol sulphate hydrolysis. The product f i r s t passes through the f i r s t (reference) c e l l and, a f t e r a l k a l i n i z a t i o n w i t h NaOH, i t passes through the second (sample) c e l l , i n which t h e c o l o u r s p e c i f i c f o r t h e arylsulphatase product i s monitored. This enzyme a l s o hydrolyses p-nitrophenol sulphate, b u t the n a t u r a l substrate f o r arylsulphatase A i s cerebroside sulphate, which accumulates i n the body i n the case o f a deficiency o f t h i s enzyme. Bostick e t a l . (1978) a l s o described the separation and a n a l y s i s o f a r y l sulphatase isoenzymes i n o t h e r human body f l u i d s . Sera from p a t i e n t s w i t h c o l o r e c t a l cancer were examined. Arylsulphatase was a l s o mentioned i n a review by Regnier and Gooding (1981), describing the r a p i d separation o f p r o t e i n s i n c l u d i n g isoenzymes f o r the purpose o f c l i n i c a l analysis. 8.3 EXAMPLES OF ENZYME SEPARATIONS 8.3.1
Proteolytic enzymes
Buchholz e t a l . (1982) used SEC on LiChrosorb D i o l t o study changes i n enzymes i n s o l u t i o n , e.g., f o r the r a p i d a n a l y s i s o f t r y p s i n a u t o l y t i c degradat i o n . Fig. 8.2 i l l u s t r a t e s t h a t the decrease i n the height o f the f i r s t chromatographic peak f o l l o w s t h e decrease i n enzymic a c t i v i t y o f the enzyme s o l u t i o n , suggesting t h a t the f i r s t peak i n HPLC corresponds t o the i n t e g r a l a c t i v e enzyme. S t r i c k e r e t a l . (1981) p u r i f i e d m i l l i g r a m amounts o f commercially prepared bovine t r y p s i n (TRY) by RP-HPLC. T i t a n i e t a l . (1982) described a simple and r a p i d p u r i f i c a t i o n o f commercial TRY and chymotrypsin (CHY) by RP-HPLC, using aceton i t r i l e i n d i l u t e t r i f l u o r o a c e t i c a c i d a t pH 2. The enzymes were prepared i n amounts appropriate f o r the s t r u c t u r a l a n a l y s i s o f proteins. Each p u r i f i e d enzyme showed the s i n g l e expected substrate s p e c i f i c i t y . Strop and Cechovd (1981) separated a- and B-trypsin by H I C on both a n a l y t i c a l and preparative scales. Gooding e t a l . (1984) described the a n a l y s i s o f p r o t e i n s w i t h new, m i l d l y hydrophobic HPLC packing m a t e r i a l s (cf., Fig. 7.6). I n another p a r t o f t h i s work, the a c t i v i t y o f TRY was monitored using post-column o n - l i n e d e t e c t i o n w i t h benzoyl-D,L-arginine
p - n i t r o a n i l i d e (BANA) ( c f .
, Section
8.2.2).
875
I
I
I
50 100 Time (min)
I
150
F i g . 8.2. A u t o l y t i c degradation o f t r y p s i n i n s o l u t i o n as a f u n c t i o n o f time, f o l l o w e d by t h e decrease i n t h e h e i g h t o f t h e f i r s t peak i n t h e HPLC t r a c e ( 7 ) and by measurement o f t h e enzymic a c t i v i t y u s i n g t h e s u b s t r a t e N-a-benzoyla r g i n i n e p - n i t r o a n i l i d e (0). ( R e p r i n t e d f r o m Buchholz e t al., 1982.) Another group o f workers used a f f i n i t y chromatography f o r t h e r a p i d i s o l a t i o n o f p r o t e o l y t i c enzymes. Kasche e t a1 , (1981) immobil i z e d soybean t r y p s i n i n h i b i t o r as a b i o s p e c i f i c adsorbent by t h e g l u t a r d i a l d e h y d e method on aminos i l a n i z e d LiChrospher. a- and 6-TRY were separated w i t h a pH g r a d i e n t and chymotrypsinogen and a-CHY were separated a t c o n s t a n t pH. The k i n e t i c s o f t h e
, Section Works , Prague,
Turkovd e t a l . (1981) used Separon H
separation were s t u d i e d ( c f .
3.9).
(Laboratory Instrument
Czechoslovakia) d e r i v a t i z e d w i t h
e p i c h l o r o h y d r i n as a support f o r l a r g e - s c a l e HPLAC. F o r t h e p r e p a r a t i o n o f a s p e c i f i c sorbent f o r c a r b o x y l i c proteinases, t h e l i g a n d E-aminocaproyl-L-PheD-Phe-OCH3 was synthesized and c o v a l e n t l y attached t o t h e support (see a1 so Section 4.4.5).
The p r o t e i n a s e s from r a w pepsin and AspergiZZus oryzae were
i s o l a t e d u s i n g t h i s s o r p t i o n m a t e r i a l . S p e c i f i c sorbents f o r high-performance l i q u i d a f f i n i t y chromatography (HPLAC) and t h e l a r g e - s c a l e i s o l a t i o n o f proteinases were a l s o d e a l t w i t h by Turkovd (1982). Small e t a l . (1981) used packings c o n t a i n i n g immobilized t r i a z i n e dyes ( c f . HPLAC o f carboxypeptidase (CP) 6-2,
, Section
4.4.5)
f o r the
i n a d d i t i o n t o o t h e r enzymes and p r o t e i n s ;
f o r t h e d e t e c t i o n o f CP t h e change i n absorbance a t 320 nm was f o l l o w e d , when methotrexate (4-amin0-N~~-methylpteroylglutamate)was hydrolysed t o 2,4-diamino10 N -methylpteroate (McCullough e t al., 1971). Marceau e t a l . (1983) described t h e r a p i d assay o f human plasma carboxypeptidase N b y HPLC s e p a r a t i o n o f h i p p u r y l
B76 l y s i n e and i t s products. Wunderwald e t a l . (1983) studied t h e removal o f endoproteinases from b i o l o g i c a l f l u i d s by "sandwich a f f i n i t y chromatography" w i t h a2-macroglobulin bound t o zinc chelated Sepharose. TRY, CHY, thermolysin, elastase, bromelain, f i c i n and papain were bound, b u t n o t exoproteinases such as carboxypeptidases A and Y. The simple loading procedure, simple regeneration and h i g h capacity are advantages o f the method (see a l s o Section 7.4.3).
Shimura
e t a l . (1984) described the HPLAC o f plasmin and plasminogen on a h y d r o p h i l i c v i n y l polymer gel (Toyopearl HW 65 S ) w i t h p-aminobenzamidine. The column packed w i t h t h i s material r e t a i n e d both plasmin and plasminogen. Plasminogen was e l u t e d w i t h 6-aminohexanoic a c i d (a haptenic compound f o r t h e l y s i n e - b i n d i n g s i t e o f plasminogen). For the e l u t i o n o f plasmin, t h e coexistence o f 6-aminohexanoic a c i d and leupeptin (a competitive i n h i b i t o r o f plasmin) was necessary. Monitoring was e f f e c t e d by f l u o r i m e t r i c d e t e c t i o n o f the e l u t e d p r o t e i n and one-line assay o f plasmin a c t i v i t y using peptidylmethylcoumarylamide, a fluorogenic substrate (cf.,
Fig. 5.18).
Gooding and Schmuck (1983) p u r i f i e d TRY and o t h e r basic p r o t e i n s (CHY, lysozyme and cytochrome c) by cation-exchange HPLC. A SynChropak CM 300 column was used and sodium acetate proved b e s t f o r i o n i c strength gradient e l u t i o n , TRY a c t i v i t y was monitored using BANA (see Section 8.2.2) reagent GPNA was used (see Section 8.2.2).
and f o r the d e t e c t i o n o f CHY the Cohen e t a l . (1984) demonstrated
m u l t i p l e peak formation i n the RP-HPLC o f papain: t h e f i r s t peak was n a t i v e and the second was p a r t o f the enzyme, denatured i n contact w i t h t h e column. 8.3.2
C e l Z u l o l y t i c , p e c t o l y t i c and amyZoZytic enzymes
Montenecourt e t a l . (1980) studied the biochemical nature o f c e l l u l a s e s from t w o physical hypercel l u l o l y t i c mutants o f Trichoderma r e e s e i using HPLC on DEAEs i l i c a (Applied Science Labs.,
State College, PA, U.S.A.).
Buchholz e t a l . (1982) c o r r e l a t e d the a c t i v i t y and the molecular s i z e o f c e l l u l a s e components using high-performance SEC on LiChrosorb Diol columns. For t h e determination o f the c e l l u l a s e a c t i v i t i e s , glucanases were analysed by incubation w i t h Avicel; samples were taken a t i n t e r v a l s , c e n t r i f u g e d and analysed f o r glucose and c e l l o b i o s e by HPLC on a LiChrosorb-NH2 (Knauer) column w i t h a c e t o n i t r i l e - w a t e r as t h e eluent and a d i f f e r e n t i a l refractometer detector. a-Glucosidases were determined by incubation w i t h c e l l o b i o s e and analysis as before. The main a c t i v i t i e s were found i n the main peaks. Hostomskd and Mikes (1983) described the a n a l y t i c a l MPLC o f c e l l u l o l y t i c enzymes on Spheron ion-exchangers and a l s o (Hostomski and Mikes, 1984) separated the c e l l u l o l y t i c system o f Trichoderma u i r i d e - r e e s e i mutant by MPLC on the l a r g e preparative scale and i s o l a t e d a new ezo-cellobiohydro1ase.
B77 MikeS e t a l . (1981) s t u d i e d t h e MPLC s e p a r a t i o n o f p e c t i c enzymes (endo-Dgalacturonase, exo-D-galacturonase,
p e c t i n l y a s e and p e c t i n esterase) from
Pectinex U1 t r a ( o r i g i n a t i n g f r o m A s p e r g i l l u s niger f e r m e n t a t i o n ) and Rohament P t e c h n i c a l p e c t o l y t i c p r e p a r a t i o n s . A l l a v a i l a b l e Spheron i o n exchangers were t e s t e d f o r t h e separation o f these p e c t i c enzymes. The experience f r o m these s t u d i e s was used f o r a s i m i l a r a n a l y s i s o f p e c t i c enzymes (Rexovd-Benkovd e t al., 1982) i n t h e Czechoslovak t e c h n i c a l p e c t o l y t i c p r e p a r a t i o n Leozym, which i s a by-product i n t h e manufacture o f c i t r i c a c i d by f e r m e n t a t i o n o f A s p e r g i l l u s n i g e r . MikeS and Rexovd (1988) reviewed techniques f o r t h e HPLC o f p e c t i c en-
zymes. Fourmy e t a l . (1982) described a r a p i d q u a n t i t a t i v e method f o r t h e d e t e c t i o n o f macroamylase i n human serum by HPLC (SEC). Isoamylases f r o m s a l i v a and p a n c r e a t i c j u i c e were a l s o analysed. Serum f r o m normal persons c o n t a i n e d two amylase peaks, d i s t a n t from t h e v o i d volume. The second peak was markedly h i g h e r i n serum from p a t i e n t s w i t h acute p a n c r e a t i t i s and mumps. I n c o n t r a s t , t h e amylase a c t i v i t y i n serum from p a t i e n t s w i t h macroamylasaemia was e l u t e d i n t h e v o i d volume. 8.3.3
Oxidoreductases
High-performance 1 i q u i d a f f i n i t y chromatography (HPLAC) has been used succ e s s f u l l y f o r t h e separation o f many enzymes. Horse 1 i v e r a l c o h o l dehydrogenase (LADH) and p i g h e a r t l a c t a t e dehydrogenase (LDH) were w e l l separated from each 6 o t h e r and f r o m bovine serum albumin on an N -(6-aminohexyl)-AMP bonded s i l i c a column. B i o s p e c i f i c e l u t i o n u s i n g d i l u t e s o l u t i o n s o f NAD+-pyrazole and NAD+pyruvate were used, i n a d d i t i o n t o a h i g h c o n c e n t r a t i o n o f sodium c h l o r i d e s o l u t i o n . When a g r a d i e n t o f NADH was applied, and M4 LDH isoenzymes (Ohlson e t a l . ,
i t was p o s s i b l e t o separate H4
1978). Lowe e t a l . (1981) used Cibacron
Blue F3G-A bonded s i l i c a f o r t h e f u l l y automated HPLAC r e s o l u t i o n o f dehydrogenases (such as LADH and LDH) , i n a d d i t i o n t o hexokinase, 3-phosphoglycerate kinase and o t h e r enzymes. Simultaneous d e t e c t i o n was achieved by m o n i t o r i n g t h e change i n absorbance a t 340 nm ( c f . ,
Section 8.2.4).
Small e t a l . (1981)
immobilized a number o f t r i a z i n e dyes t o m i c r o p a r t i c u l a t e s i l i c a and s t u d i e d t h e i r a p p l i c a t i o n f o r t h e s e p a r a t i o n o f LDH and o t h e r enzymes. B o r c h e r t e t a1
.
(1982) immobilized concanavalin A t o porous s i l i c a and used t h i s support f o r t h e a n a l y s i s and p u r i f i c a t i o n o f glucose oxidase and g l u c o p r o t e i n s peroxidase. This HPLAC method was s t u d i e d i n d e t a i l . I E X has a l s o o f t e n been used f o r t h e r a p i d s e p a r a t i o n o f enzymes. Matsumoro (1981) described t h e t h e o r y and use o f serum LDH isoenzyme a n a l y s i s on I E X 525
878
QAE; w i t h a gradient of NaCl concentration t h e t o t a l separation o f isoenzymes was achieved w i t h i n 20 min. Van der Wal and Huber (1980) studied t h e performance of c l a s s i c a l n o n - r i g i d ion-exchange packings o f small p a r t i c l e s i z e i n the separation of cytochrome c and d e r i v a t i v e s by HPLC, b u t b e t t e r r e s u l t s were obtained w i t h (meth)acryl i c c a t i o n exchangers and hydroxyapatite. Vanecek and Regnier (1982) described a r a p i d separation o f lipoxygenase I using a column o f LiChrospher S i 4000 coated w i t h a heavy l a y e r o f polyethylenimine and crossl i n k e d w i t h 1,4-butanediol
d i g l y c i d y l ether. Lindblom (1983) developed a simple
method f o r the i s o l a t i o n o f glucose-6-phosphate dehydrogenase from a yeast enzyme concentrate. A column o f Polyanion S I (8 pm) was used f o r a n a l y t i c a l and Polyanion S I (17 pm) f o r preparative purposes. Hearn e t a l . (1980) studied the SEC o f sheep l i v e r aldehyde dehydrogenase (and t h y r o g l o b u l i n ) on pBondage1 E-linear. I n d i c a t i o n s o f the i n f l u e n c e o f a mixed-mode separation p r i n c i p l e were found. Power e t a l . (1983) used RP-HPLC f o r the p u r i f i c a t i o n o f nuclear coded subunits o f a membrane oligomeric p r o t e i n
-
yeast cytochrome c oxidase. 8.3.4 Enzymes of phosphate metaboZism, other miscellaneous enzymes and enzymic
reactions Small e t a l . (1981) used dye-ligand chromatography f o r the f r a c t i o n a t i o n o f yeast hexokinase on Procion Green H-4G s i l i c a , and of L-tryptophanyl - t R N A synthetase o f Cibacron Blue F3G-A s i l i c a and Procion Brown MX-5BR s i l i c a . Lowe e t a l . (1981) studied chromatography on Cibacron Blue FSG-A s i l i c a and detection methods f o r hexokinase, 3-phosphoglycerate kinase ( c f . , Section 8.2.4)
and a l s o
o f pancreatic ribonuclease A. Mikes e t a l . (1978) described t h e r a p i d I E C separation o f technical enzymes k.g.,
crude protease from AspergiZZus sojae and glucose oxidase from Aspergiltus
niger (Fig. 8.3)]. Kato e t a l . (1980) p u r i f i e d crude 8-galactosidase from b a c t e r i a l c e l l s and commercial urease by SEC on TSK Gel 3000 SWG. Roughly 15-fold p u r i f i c a t i o n was achieved i n a s i n g l e f i l t r a t i o n . Lim (1979) developed HPLC methods f o r the determination o f enzymes o f the haeme b i o s y n t h e t i c pathway (6-aminolaevul i n i c a c i d synthetase and dehydratase, uroporphyrinogen I synthetase). The enzymes were n o t isolated, b u t determined by the separation of t h e i r low-molecular-weight products. Studebaker (1979) reviewed the analysis o f enzymic reactions by HPLC.
879
25
a
0 I
I
10
20
I
30
F.N.
Fig. 8.3. Chromatography of technical glucose oxidase on a DEAE-Spheron column (20 crn x 0.8 cm). Load: 15 mg of preparation in 0.2 m l of buffer A. The ion exchanger was e q u i l i b r a t e d w i t h b u f f e r A. I , Buffer A without g r a d i e n t . Linear gradients I1 ( A + B ) and I11 ( B + C). IV, buffer C without g r a d i e n t . Flow-rate, 2 ml/min; 4-ml f r a c t i o n s ; temperature, 14oC; counter pressure, 3-7 atm; c h a r t speed, 2 mm/min. Buffers: A, 0.01 M a c e t i c a c i d + NaOH, pH 6.8; B y 0.3 M a c e t i c acid + NaOH, pH 5.5; C , b u f f e r B y 1 M in NaC1, pH 5.3. Broken l i n e , glucose oxidase a c t i v i t y of e f f l u e n t i n S a r r e t u n i t s (S.U.; 1 S.U. corresponds t o t h e consumption of 600 p1 of oxygen a t 30oC). F.N. = Fraction number; M = automatic marking of f r a c t i o n c o l l e c t i o n ; mS = e l e c t r i c conductivity in mS. Corresponding peaks in both p a r t s o f t h e chromatogram a r e designated by a - i . The UV spectrum of the chromophore o f compound d d i f f e r e d s i g n i f i c a n t l y from t h e others. (Reprinted from Mike: e t a l . , 1978.) 8.4 COMMENTS ON LITERATURE G u i l b a u l t ' s (1976) Handbook i s not devoted t o HPLC methods, b u t i t describes in d e t a i l assay procedures f o r 39 important enzymes, enzymic analyses of more than 55 s u b s t r a t e s and t h e a p p l i c a t i o n of immobilized enzymes in enzymic analyses, and provides much additional p r a c t i c a l information. Regnier e t a l . (1979) and Regnier and Gooding (1981) reviewed t h e HPLC of p r o t e i n s , including t h e HPLC of enzymes; these reviews cover specialized a p p l i c a t i o n s in c l i n i c a l a n a l y s i s . Ishiguro and Shinohara (1981) reviewed t h e use of IEC, SEC, a f f i n i t y , adsorption and high-performance 1 iquid chromatography in the separation o f isoenzymes (especially of l a c t a t e dehydragenase, c r e a t i n e phosphate kinase, glutamate-
B80
o x a l a c e t a t e transaminase, a1 k a l i n e phosphatase and ma1 i c dehydrogenase). Mikes
(1981/1982 and 1982) d e a l t w i t h t h e r a p i d chromatographic a n a l y s i s o f enzymes and o t h e r p r o t e i n s f o r a p p l i c a t i o n t o f o o d c h e m i s t r y . Vacik and Toren (1982) reviewed t h e s e p a r a t i o n and measurement o f isoenzymes and o t h e r p r o t e i n s by HPLC; t h i s t r e a t i s e covered p r e d o m i n a n t l y b i o m e d i c a l a p p l i c a t i o n s . Enzyme s e p a r a t i o n by RP-HPLC was reviewed by S t r i c k l e r e t a l . (1984). Jakoby
(1984) e d i t e d a s p e c i a l volume o f Methods in EnzymoZogy d e a l i n g w i t h t h e p u r i f i c a t i o n o f enzymes, where HPLC t e c h n i q u e s f o r enzyme s e p a r a t i o n s were d e s c r i b e d i n d e t a i l ; Wehr d e a l t w i t h t h e c a r e o f HPLC columns, Unger e x p l a i n e d SEC methods, Regnier I E C , Richey o p t i m a l pH c o n d i t i o n s f o r I E C , Hearn RPC and Larsson HPLAC.
REFERENCES Aoshima, H., Anal. Biochem., 95 (1979) 371-376. Bender, M.L. , Begue-Canton, M.L. , B l a k e l y , R.L. , Brubacher, L.J. , Feder, J., Gunter, C.R., Kezdy, F.J. , K i l l h e f f e r , J.V., M a r s h a l l , T.H. , M i l l e r , C.G. , Roeske, R.W. and Stoops, J.K., J . Am. Chem. Soc., 88 (1966) 5890-5913. B o r c h e r t , A,, Larsson, P.-0. and Mosbach, K., J . Chromatogr., 244 (1982) 49-56. B o s t i c k , W.D., Denton, M.S. and Dinsmore, S.R., CZin. Chem., 26 (1980) 712-717. B o s t i c k , W.D. , Dinsmore, S.R. , Mrochek, J.E. and Waal kes, T.P. , CZin. Chem., 24 (1978) 1305-1316; C.A., 89 (1978) 142 3709. Buchholz, K., Gijdelmann, B. and Molnar, I.,J . Chromatogr., 238 (1982) 193-202. Chang, S.H., Gooding, K.M. and Regnier, F.E., J . Chromatogr., 125 (1976) 103-114. Chase, T. and Shaw, E., Biochem. Biophys. Res. C o m n . , 29 (1967) 508-514. Cohen, L., D j o r d j e v i c h , J. and Ormiste, V., J . Lab. Clin. Med., 64 (1964) 355374. Cohen, S.A., Benedek, K.P., Dong, S.T.Y. and Karger, B.L., AnaZ. Chem., 56 (1984) 217-221. Denton, M.S., B o s t i c k , W.D. and Dinsmore, S.R., CZin. Chem., 24 (1978) 1408-1413; C.A., 89 (1978) 142 373k. Denton, M.S., B o s t i c k , W.D. , Dinsmore, S.R. and Mrochek, J.E., BioZogicaZ/BiornedicaZ Applications of Liquid Chromatography 2 (Chrorratographic Science S e r i e s , Vol. 12), Marcel Dekker, New York, 1979, pp. 165-191; C . A . , 92 (1980) 36 513h. E l k i n s , B.N., CZin. Chem., 23 (1977) 1510. Evrev, T.I., i n M a r k e r t , C. ( E d i t o r ) , Isoenzymes, Vol. 2, Academic Press, New York, 1975, p. 129. Fourmy, D., P r a d a y r o l , L. , Bommelaer, G. and R i b e t , A., Gastroenterol. C l i n . BioZ., 6 (1982) 249-251; C.A., 96 (1982) 176 658s. F u l t o n , J.A., Schlabach, T.D., K e r l , J.E. and Toren, E.C., J . Chromatogr., 175 (1979) 283-291. Galen, R.S. and Raymond, G.S., C l i n . Chem., 21 (1975) 1848-1850; C.A., 84 (1976) 15 416y. Galen, R.S., R e i f f e l , J.A. and Gambino, S.R., J . Am. Med. Assoc., 232 (1975) 145. Gooding, D.L., Schmuck, M.N. and Gooding, K.M., J . Chromatogr., 296 (1984) 107114. Gooding, K.M. and Schmuck, M.N., J . Chromatogr., 266 (1983) 633-642. G u i l b a u l t , G.G., Handbook of Enzymatic Methods of Analysis (CZinicaZ and Biochemical, Analysis, Vol. 41, Marcel Dekker, New York, Basle, 1976, 738pp. Haverback, B.J., Dyce, B., Bundy, H. and Edmonson, H.A., Am. J . Med., 29 (1960) 424. Hearn, M.T.W., Methods EnzymoZ., 104C (1984) 190-212.
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Hearn, M.T.W., Grego, B., Bishop, C.A. and Hancock, W.S., J . Liq. Chromatogr., 3 (1980) 1540-1560. Hostomskd, Z. and Mikes, O., J . Chromatogr., 267 (1983) 355-366. Hostomskd, Z. and Mikes, O., I n t . J . Peptide Protein Res., 23 (1984) 402-410. I n t e r n a t i o n a l Union of B i o c h e m i s t r y ( I U B ) , Enzyme NornencZature, E l s e v i e r , Amsterdam, 1965, 1972. I s h i g u r o , I. and Shinohara, R., Rinsho Kensa, 25 (1981) 183-188; C.A., 94 (1981) 170 153k. Jakoby, W.B. ( E d i t o r ) , Enzyme & r i f i c a t i o n and Related Techniques (Methods Enzymol., Vol. 104C), Academic Press, New York, 1984, 528 pp. Kasche, V., Buchholz, K. and Galunsky, B., J . Chromatogr., 216 (1981) 169-174. Kato, Y . , Komiya, K., Sawada, Y. , Sasaki, H. and Hashimoto, T., J . Chromatogr., 190 (1980) 305-310. Kudirka, P.J., Bushy, M.G. and Toren, E.C., Jr., CZin. Chem., 21 (1975) 450-452. K u d i r k a , P.J., Schroeder, R.R., H e w i t t , T.E. and Toren, E.C., Jr., CZin. Chem., 22 (1976) 471-474. . Larsson, P . 4 . , Methods Enzymol. , 104C (1984) 212-223. Lim, C.K., Proc. Anal. Div. Chem. Soc.. 16 (1979) 305-307; C.A.. 92 (1980) ii 551;. Lindblom, H., J . Chromatogr., 266 (1983) 265-281. Lowe, Ch.R. , Glad, M. , Larsson, P.-0. , Ohlson, S., Small, D.A.P. , A t k i n s o n , T. and Mosbach, K., J . Chromatogr., 215 (1981) 303-316. Marceau, F., D r u m h e l l e r , A., Gendreau, M., L u s s i e r , A. and S t - P i e r r e , S., J . Chromatogr., 266 (1983) 173-177. Matsumoto, K., Kensa t o G i j u t s u , 9 (1981) 359-366; C.A., 94 (1981) 187 5 1 0 ~ . McCullough, J.L., Chabner, B.A. and B e r t i n o , J.R. , J . Biol. Chem., 246 (1971) 7207-7213. M e i s t e r , A., J . BioZ. Chem., 184 (1950) 117-129. Mikes, O., EmGhmtng/Nutrition, 5 (1981) 88-98 ( i n E n g l i s h , r e f e r e n c e s ommited); C . A . , 94 (1981) 152 760b. Czech t r a n s l a t i o n w i t h f u l l r e f e r e n c e s : Chem. L i s t y , 76 (1982) 59-79; C.A., 96 (1982) 118 2 9 7 ~ . Mikes, 0. and Rexovd, L ' . , Methods Enzymol., 161B (1988) 385-399. Mikes, 0. , i n B a l t e s , W. , Czedik-Eysenberg, P.B. and Pfannhauser, W. ( E d i t o r s ) , Recent Developments i n Food AnaZysis (Proceedings o f t h e 1 s t European Conf e r e n c e on Food Chemistry, EURO FOOD CHEM I , Vienna, A u s t r i a , February 1981), V e r l a g Chemie, Weinheim, 1982, pp. 306-321. Mikes, O., i n Deyl, Z., Macek, K. and Jandk, J. ( E d i t o r s ) , Liquid CoZwrm Chromatography, A Survey of Modern Techniques and Applications (Journal of chromatography Library, Vol 3), E l s e v i e r , Amsterdam, 1975, pp. 807-830. Mike5, O., SedldEkovd, J., Rexovd-Benkovd, L. and Omelkovd, J. , J . Chromatogr., 207 (1981) 99-114. Mikes, O . , S t r o p , P. and SedldEkovd, J., J . Chromatogr., 148 (1978) 237-245. Montenecourt, B.S. , K e l l e h e r , T.J., E v e l e i g h , D. and P e t t e r s s o n , L.G., Biotechnol. Bioeng. Symp., 10 (1980); C . A . , 94 (1981) 26 6 3 1 ~ . Morin, L.G., C l i n . Chem., 23 (1977) 205-210. N e i l a n d s , J.B., Science (Washington, D . C . ) , 115 (1952) 143-144. Ohlson, S., Hansson, L., Larsson, P.-0. and Mosbach, K., FEBS L e t t . , 93 (1978) 5-9. Power, S.D., L o c h r i e , M.A. and Poyton, R.O., J . Chromatogr., 266 (1983) 585-598. Pressey, R., HortScience, 19 (1984) 572-573. P r u s i k , Z., i n Deyl, Z., Macek, K. and Jandk, J. ( E d i t o r s ) , Liquid CoZwrm Chromatography, A Survey of Modern Techniques and Applications, (Journal of Chromatography Library, V o l . 3), E l s e v i e r , Amsterdam, 1975, p. 803. Raschbaum, G.R. and Everse, J., Anal. Biochem., 90 (1978) 146-154. Regnier, F.E., Methods EnzymoZ., 104C (1984) 170-189. Regnier, F.E. and Gooding, K.M., i n Laurence, J., Marton, J . and Kabra, P.M. ( E d i t o r s ) , Liquid Chromatography i n CZinicaZ Analysis, Humana Press , C1 i f t o n , NJ, 1981, pp. 323-353.
.
Regnier, F.E., Gooding, K.M. and Chang, S.H., Contemp. Top. Anal. Chem., 1 (1977) 1-48; C.A., 87 (1977) 196 576f. Remy, M.H., Guillochon, D. and Thomas, D., J . Chromatogr., 215 (1981) 87-91. Rexovd-Ben kovd , L , Omel kovd , J. , M i keZ , 0. and Sedl dEkovd , J , J . Chromatogr. , 238 (1982) 183-192. Richey, J.S. , Methods Enzymol. , 104C (1984) 223-233. Schlabach, T.D., A l p e r t , A.J. and Regnier, F.E., Clin. Chem., 24 (1978) 13511360. Schlabach, T.D. and Regnier, F.E., J . Chromatogr., 158 (1978) 349-364. Schlabach, T.D. , Chang, S.H. , Gooding, K.M. and Regnier, F.E. , J . Chromatogr. , 134 (1977) 91-106. Schlabach, T.D., Fulton, J.A., Mockridge, P.B. and Toren, E.C., Jr., AnaZ. Chem., 52 (1980a) 729-733. Schlabach, T.D., F u l t o n , J.A., Mockridge, P.B. and Toren, E.C., Jr., C Z i n . Chem., 25 (1979) 1600-1608. Schlabach, T.D., Fulton, J.A., Mockridge, P.B. and Toren, E.C., Jr., Clin. Chem., 26 (1980b) 707-711; C.A., 93 (1980) 21 320q. Schroeder, R.R., Kudirka, P.J. and Toren, E.C., Jr., J . Chromatogr., 134 (1977) 83-90, Shimura, K., Kazama, M. and Kasai, K.-I., J . Chromatogr., 292 (1984) 369-382. Atkinson, T. and Lowe, Ch.R., J . Chromatogr., 216 (1981) 175-190. Small, D.A.P., S t r i c k l e r , M.P. and Gemski, M.J., i n Hancock, W.S. ( E d i t o r ) , CRC Handbook of HPLC f o r the Separation of Amino Acids, Peptides, and Proteins, V O ~ 11, CRC Press, Boca Raton, FLY 1984, pp. 349-355. S t r i c k l e r , M.P., Gemski, M.J. and Doctor, B.P., J . Liq. Chromatogr., 4 (1981) 1765-1 775. Strop, P. and Eechovd, D., J . Chromatogr., 207 (1981) 55-62. Studebaker, J.F., Biological/BiomedicaI Ap l i c a t i o n s o f Liquid Chromatography 1 (Chromatography Science Series, Vol. 107, Marcel Dekker, New York, 1979, pp. 261-281; C.A., 90 (1979) 147 426k. T i t a n i , K., Sasagawa, T., Resing, K. and Walsh, K.A., AnaZ. Biochem., 123 (1982) 408-412. Visser, J. and Nivard, R.J.F. ( E d i t o r s ) , Turkovd, J., i n Gribnau, T.C.J., A f f i n i t y Chromatography and Related Techniques, E l s e v i e r , Amsterdam, 1982 , pp. 513-528. Turkovd, J., Bldha, K., HordEek, J., VajEner, J., Frydrychovd, A. and Coupek, J., J . Chromatogr., 215 (1981) 165-179. Unger, K. , Methods k z y m o l . , 104C (1984) 154-169. Vacik, D.N. and Toren, E.C., Jr., J . Chromatogr., 228 (1982) 1-31. Van den Wal, S j . and Huber, J.F.K., Anal. Biochem., 105 (1980) 219-229. Vanecek, G. and Regnier, F.E., AnaZ. Biochem., 121 (1982) 156-169. Vesell, E.S. and Bearn, A.G., Proc. Soc. Exp. B i o l . Med., 94 (1957) 96-99; C.A., 51 (1957) 10 606a. Wehr, C.T. , Methods Enzymol., 104C (1984) 133-154. Wunderwald, P., Schrenk, W.J., P o r t , H. and Kresze, G.-B., J . A p p l . Biochem., 5 (1983) 31-42.
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Chapter 8 ENZYMES 8.1 I I ~ T R O D U C T I O N
Enzymes are p r o t e i n s and t h e r e f o r e the methods and problems described and discussed i n Chapter 7 are v a l i d a l s o f o r enzymes. However, t h e r e a r e some special features t h a t must be emphasized when the separation o f enzymes i s considered. I n general, enzymes are o f t e n very s e n s i t i v e t o denaturation, u s u a l l y r e s u l t i n g i n a t o t a l o r p a r t i a l l o s s o f c a t a l y t i c a c t i v i t y . The y i e l d o f an enzyme f r a c t i o n a t i o n must be expressed n o t o n l y i n terms o f t h e p r o t e i n recovery, b u t a l s o i n terms o f the recovery o f enzymic a c t i v i t y . The increase i n the r a t i o of the a c t i v i t y t o the p r o t e i n content i s the usual measure o f the degree o f p u r i f i c a t i o n i n the process o f enzyme i s o l a t i o n . The a c t i v i t y / p r o t e i n r a t i o i s a1 so an important constant c h a r a c t e r i z i n g the pure and homogeneous enzyme preparation. The r i s k o f denaturation o f enzymes a r i s e s n o t o n l y w i t h elevated temperature. F r a c t i o n a t i o n a t temperatures several degrees above zero i s u s u a l l y used n o t only t o e l i m i n a t e the p o s s i b i l i t y o f thermal denaturation o f enzymes, b u t a l s o because many raw m a t e r i a l s o f b i o l o g i c a l o r i g i n contain c e r t a i n amounts o f various proteinases, which can p a r t l y decompose the enzymes t o be i s o l a t e d , e s p e c i a l l y a t the usual ambient temperatures. Unknown enzyme preparations should c e r t a i n l y n o t be exposed t o extreme c o n d i t i o n s o f pH, which very e a s i l y change t h e i r t e r t i a r y structure. Organic solvents represent another t h r e a t t o the a c t i v i t y o f some enzymes e s p e c i a l l y a t elevated temperatures. This must be considered when reversed-phase high-performance 1 i q u i d chromatography (RP-HPLC) experiments a r e planned. The same applies t o detergents: sodium dodecylsulphate s o l u t i o n i s useful f o r t h e determination o f molecular weights of enzymes using t h e s i z e exclusion chromatography (SEC) mode, b u t t h e a c t i v i t y i s destroyed. Some enzymes form special quaternary s t r u c t u r e s and the detergents d i s s o c i a t e t h i s s t r u c t u r e i n t o i n d i vidual subunits. Other surface-active compounds a l s o endanger enzymes. Many enzymes cannot t o l e r a t e c o n t a c t w i t h ions o f heavy metals, and t h i s must be borne i n mind when b u f f e r s a r e selected f o r t h e e l u t i o n o f enzymes from s t a i n less-steel chromatographic columns: some b u f f e r s a t t a c k the s t a i n l e s s s t e e l (e.g.,
a c i d i c s o l u t i o n s o f ammonium formate) and t r a c e amounts o f heavy metal
B64 i o n s can d e s t r o y t h e enzyme a c t i v i t y i n some i n s t a n c e s ; g l a s s o r o t h e r nonc o r r o s i v e columns and pumps a r e t o be p r e f e r r e d . Oxygen i n t h e a i r ( a l s o d i s s o l v e d i n t h e m o b i l e phase) can d e a c t i v a t e some s e n s i t i v e enzymes. I n p a r t i c u l a r , t h e enzymes o f anaerobic microbes and many o t h e r enzymes o f anaerobic metabolism may be s e n s i t i v e t o oxygen t o such an e x t e n t t h a t t h e a d d i t i o n o f r e d u c i n g agents t o t h e m o b i l e phase i s n o t s u f f i c i e n t t o p r o t e c t t h e a c t i v i t y and t h e s e p a r a t i o n must be performed i n a s p e c i a l anaero b i c l a b o r a t o r y . S t r o n g r e d u c i n g agents w i l l a l s o d e s t r o y t h e a c t i v i t y o f some enzymes. S u r f a c e d e n a t u r a t i o n o f enzymes i n c o n t a c t w i t h u n s u i t a b l e chromatographic sorbents t h a t a l l o w s t r o n g hydrophobic i n t e r a c t i o n s has o f t e n been observed; h y d r o p h i l i c adsorbents a r e p r e f e r a b l y used. F o r t h i s reason, t h e n e w l y developed h y b r i d h y d r o p h i 1 i c / h y d r o p h o b i c pac k i n g s f o r hydrophobic i n t e r a c t i o n c hromatography (HIC) o f enzymes a r e v e r y i m p o r t a n t ( c f . ,
S e c t i o n s 3.6 and 4.4.3).
The t o t a l l o s s o f enzymic a c t i v i t y due t o t h e d e n a t u r a t i o n o f n a t i v e enzymes i s n o t t h e o n l y r i s k when enzymes a r e separated. What may be n e g l e c t e d a r e s l i g h t changes i n c o n f o r m a t i o n , o r 1 i m i t e d p r o t e o l y s i s , which s l i g h t l y m o d i f y enzymes so t h a t t h e y d i f f e r i n t h e i r chromatographic r e t e n t i o n f r o m t h e o r i g i n a l n a t i v e form, b u t s t i l l r e t a i n t h e i r a c t i v i t y . A r t i f i c i a l enzyme polymorphism i s c r e a t e d i n t h i s way and i n s t e a d o f n a t i v e enzymes t h e a c t i v e a r t i f a c t s may be i s o l a t e d . " A r t i f i c i a l isoenzymes" a r e formed t h a t can h a r d l y be d i s t i n g u i s h e d f r o m n a t u r a l isoenzymes a t t h e b e g i n n i n g o f t h e s t u d y because m u l t i p l e forms o f enzymes v e r y o f t e n o c c u r i n n a t u r e . S p e c i a l r e q u i r e m e n t s f o r t h e low-speed chromatography o f enzymes were d i s cussed f r o m t h i s p o i n t o f view, e.g.,
by Mikes (1975).
Enzymes a r e b i o c h e m i c a l c a t a l y s t s and t h e i r a c t i v i t i e s have been measured by many workers i n v a r i o u s ways. I t was e x t r e m e l y d i f f i c u l t t o compare r e s u l t s r e p o r t e d on t h e same enzyme. Hence t h e F i f t h I n t e r n a t i o n a l Congress o f Biochemi s t r y adopted t h e recommendations o f t h e Commissions on Enzymes o f I U P A C ( I n t e r n a t i o n a l Union o f Pure and A p p l i e d C h e m i s t r y ) and IUB ( I n t e r n a t i o n a l Union o f B i o c h e m i s t r y ) (1965, 1972) f o r t h e d e f i n i t i o n o f an enzyme u n i t . The recommended terms w i l l be b r i e f l y p r e s e n t e d h e r e a c c o r d i n g t o G u i l b a u l t (19761, as follows. One unit l U ) of any enzyme i s t h a t amount which w i l l c a t a l y s e t h e t r a n s f o r m a t i o n o f 1 vmol o f s u b s t r a t e p e r m i n u t e ; where more t h a n one bond o f each s u b s t r a t e i s a t t a c h e d , t h e t r a n s f o r m a t i o n o f 1 uequiv. of t h e group concerned p e r m i n u t e i s considered, under d e f i n e d c o n d i t i o n s . Recommended parameters: temperature, 25OC; pH, o p t i m a l ; r e a c t i o n r a t e , i n i t i a l ; and k i n e t i c s w i t h r e s p e c t t o t h e s u b s t r a t e , zero o r d e r .
865
Concentration o f an enzyme i n s o l u t i o n : u n i t s p e r m i l l i l i t r e (U/ml) o r p e r l i t r e (U/l).
S p e c i f i c a c t i v i t y o f an enzyme: u n i t s o f enzyme p e r m i l l i g r a m o f p r o t e i n (U/mg p r o t e i n ) .
Molecular a c t i v i t y : u n i t s p e r micromole o f enzyme, i.e.,
t h e number o f mole-
c u l e s o f t h e s u b s t r a t e t r a n s f o r m e d p e r m i n u t e p e r m o l e c u l e o f t h e enzyme. 107 m e k a t a l r e f e r s t o t h e c o n v e r s i o n o f 1 mol o f s u b s t r a t e p e r second = 6 7 pmol o f s u b s t r a t e p e r m i n u t e = 6 10 u n i t s (one u n i t = 1 U = 16.67 n k a t a l ) . PRINCIPLES OF SPECIFIC DETECTION METHODS FOR ENZYMES AND ISOENZYMES
8.2.
One o f t h e s i m p l e s t approaches i s t o measure t h e absorbance i n t h e long-wave
UV r e g i o n ( P r u s i k , 1975), which i s used f o r t h e s e n s i t i v e d e t e c t i o n o f some enzymes w i t h f i r m l y bound p r o s t h e t i c groups. However, when d e a l i n g w i t h t h e f r a c t i o n a t i o n o f c r u d e m i x t u r e s o f p r o t e i n s , s p e c i f i c enzymes must b e quant i t a t e d by enzyme assay o f f r a c t i o n s r a t h e r t h a n by s p e c t r o p h o t o m e t r i c m o n i t o r i n g o f t h e e f f l u e n t . The chromatographic column e f f l u e n t can be analysed u s i n g an o f f - l i n e o r an o n - l i n e procedure. F o r t h e o f f - l i n e procedures a l i q u o t s o f t h e f r a c t i o n s a r e t a k e n m a n u a l l y o r by some a u t o m a t i c equipment (see, e.g.,
a short
r e v i e w i n an essay by Mikes, 1975). Raschbaum and Everse (1978) a l s o d e s c r i b e d an apparatus f o r t h e a u t o m a t i c d e t e c t i o n o f enzyme a c t i v i t y i n column chromatog r a p h i c e f f l u e n t s , which c o n s i s t s o f a s i m p l e s t o p p e d - f l o w i n s t r u m e n t , equipped w i t h a m u l t i - s h u t o f f v a l v e t o d i r e c t t h e r e a g e n t and e l u e n t f l o w s : assays o f t h e e l u a t e can be c a r r i e d o u t a t p r e - s e t i n t e r v a l s . The chemical p r i n c i p l e s and a n a l y t i c a l procedures f o r t h e q u a n t i t a t i o n o f many i m p o r t a n t enzymes were d e s c r i b e d , e.g.,
i n t h e handbook by G u i l b a u l t (1976).
From t h e p o i n t o f v i e w o f t h e p r e s e n t s t a t e o f e v o l u t i o n o f t h e l i q u i d c h r o matography o f enzymes, o f f - l i n e d e t e c t i o n procedures seem t o be u s e f u l o n l y f o r p i o n e e r works i n a t t e m p t s t o i s o l a t e an unknown enzyme, o r w i t h some s i n g l e a c t i o n p r e p a r a t i v e experiments i n o r d e r t o i s o l a t e a known r e q u i r e d enzyme, and never f o r r o u t i n e l y r e p e a t e d a n a l y t i c a l o r p r e p a r a t i v e procedures. When HPLC s e p a r a t i o n s o f p r o t e i n s i n 10 min became p o s s i b l e , time-consuming o f f - l i n e enzymic assays o f t h e components were t h e l i m i t i n g s t e p . T h e r e f o r e , r a p i d o n - l i n e d e t e c t i o n methods f o r enzymes and isoenzymes were sought. The i n s t r u m e n t a t i o n o f d e t e c t o r s developed f o r t h i s purpose was d e a l t w i t h i n d e t a i l i n S e c t i o n 5.2.3.
I n t h e f o l l o w i n g sections the biochemical p r i n c i p l e s o f t h e s p e c i f i c
d e t e c t i o n methods w i l l be e x p l a i n e d . T h i s approach was e s p e c i a l l y i m p o r t a n t i n c l i n i c a l a p p l i c a t i o n s f o r t h e i d e n t i f i c a t i o n o f isoenzymes. M e i s t e r (1950) and N e i l a n d s (1952) were t h e f i r s t t o r e c o g n i z e m u l t i p l e forms o f t h e enzyme l a c t a t e dehydrogenase ( L D ) . Vessel
666 and Bearn (1957) opened a new dimension i n c l i n i c a l diagnosis by t h e i r f i n d i n g t h a t the serum p r o f i l e of LD isoenzymes changes d r a m a t i c a l l y w i t h myocardial i n f a r c t i o n o r acute myelogenous leukaemia. Cohen e t a l . (1964) described i n d e t a i l serum LD patterns i n cardiovascular and other diseases, w i t h p a r t i c u l a r reference t o acute myocardial i n f a r c t i o n . The diagnostic importance o f isoenzymes has been evaluated i n many papers (e.g.,
Galen e t al., 1975). Usually, isoenzymes
were separated from each other using mainly e l e c t r o p h o r e t i c , ion-exchange, immunochemical and various s o r p t i o n techniques. Morin (1977) evaluated the methods c u r r e n t a t t h a t time f o r c r e a t i n e kinase isoenzyme f r a c t i o n a t i o n . Kudirka e t a l . (1975) were the f i r s t t o apply modern r a p i d HPLC separation t o c r e a t i n e kinase isoenzymes, b u t the o n - l i n e detector was missing. However, the authors discussed i t s importance.
A new p o s s i b i l i t y f o r the development o f serum isoenzyme p r o f i l i n g was opened w i t h the i n t r o d u c t i o n o f HPLC methods i n t o p r o t e i n chemistry and biochemistry. The speed o f the separation i n i t i a t e d a search f o r an equivalent r a p i d o n - l i n e detection method. The d i a g n o s t i c a l l y important isoenzymes were separated from each other on HPLC columns, b u t were n o t separated from various a d d i t i o n a l serum proteins , so t h a t detection methods were necessary t h a t would be able t o "see" the isoenzymes b u t n o t the other proteins covering t h e i r peaks. The biochemical p r i n c i p l e s of such techniques a r e described i n the f o l l o w i n g sections. A l k a l i n e phosphatase
8.2.1
The s p e c i f i c detection o f a l k a l i n e phosphatase (AP) represents t h e f i r s t simple example on which on-line post-column detection can be explained (Chang e t al.
,
1976; Schlabach e t a l .
, 1977;
Schlabach and Regnier, 1978). The a n a l y t -
i c a l column e f f l u e n t i s mixed w i t h the substrate p-nitrophenyl phosphate (NPP) i n the equipment i l l u s t r a t e d i n Fig. 5.29, and the mixture i s heated (4OoC) i n the packed column reactor i n which t h e product p-nitrophenol (NP) i s formed according t o the equation o2NC6H4oPo3Na2 NPP
AP
H20
02NC6H40H + HOP03Na2 NP
The product (NP) can be r e a d i l y detected i n a l k a l i n e s o l u t i o n by measurement o f the absorbance a t 400 o r 410 nm, and the presence o f proteins (Amax 280-285 nm) has no effect. An example o f such an analysis i s i l l u s t r a t e d i n Fig. 5.30. I n a d d i t i o n t o the s e l e c t i v i t y , t h i s detection p r i n c i p l e i s more than 20 times more s e n s i t i v e than conventional absorbance monitoring o f p r o t e i n s a t 280 nm.
If a wider r e a c t o r column (250 x 8 mm 1.0.) was used and the temperature increased t o 6OoC, t h e detection l i m i t was 25 ng/ml o f a l k a l i n e phosphatase (Regnier e t a l . , 1977). 8.2.2
m p s i n and chymotrypsin
Another simple reaction s u i t a b l e f o r on-line post-column detection i s the spectrophotometric determination of trypsin (TRY), studied f o r t h i s purpose by Schlabach e t a l . (1977). Bender e t a l . (1966) described a number of stoichiometr i c reagents t h a t were developed f o r t h e t i t r a t i o n o f a c t i v e s i t e of enzymes and made i t possible t o determine the concentration o f an enzyme in solution with an end-point reaction. A very e f f e c t i v e a c t i v e s i t e t i t r a n t f o r TRY i s n i t r o phenyl guanidinoboenzoate (NPGB) (Chase and Show, 1967), which r e a c t s with TRY according t o t h e equation
H2N\//
NHbHCI
+
TRY-
The p-nitrophenol ( N P ) product can be monitored by spectrophotometry a t 400 nm. NPGB r e a c t s only with a c t i v e TRY and i s i n s e n s i t i v e t o o t h e r enzymes and t o i n a c t i v e TRY. Schlabach e t a l . (1977) demonstrated t h e l i n e a r i t y of t h e response and the s u i t a b i l i t y of the method f o r q u a n t i t a t i n g TRY in a flow-through r e a c t o r . The addition of NaCl a t a 0.5 M concentration t o the measured solution was recommended in order t o suppress t h e adsorption o f TRY on the equipment surfaces.
868 Another post-column o n - l i n e detection method f o r TRY was pub1 ished by Gooding e t a l . (1984). The e x i t from the a n a l y t i c a l HI-HPLC column was connected, using a union tee, w i t h the substrate pump and a packed column r e a c t o r containing SynChropak PCR (a m i c r o p a r t i c u l a t e non-porous packing). The e f f l u e n t from t h e reactor was monitored a t 410 nm. The substrate f o r TRY pumped t o the union t e e was benzoyl-D,L-arginine
p - n i t r o a n i l i d e (BANA) , dissolved i n 0.1 M potassium
phosphate s o l u t i o n (pH 7 ) . BANA i s hydrolysed t o p - n i t r o a n i l i n e (NA) by TRY (Hoverbach e t al.,
1960) according t o the equation
BANA
/NH C=NH
\
NH2
This i s a normal t r y p t i c c a t a l y s i s and n o t a s t o i c h i o m e t r i c r e a c t i o n as was the case i n the first-mentioned equation. A s i m i l a r method was used by Gooding and Schmuck (1983) f o r the post-column
on-1 i n e monitoring o f chymotrypsin (CHY).
Glutaryl-L-phenylalanine p - n i t r o -
a n i l i d e (GPNA) was used as a s p e c i f i c substrate, which i s hydrolysed by chymot r y p s i n t o p - n i t r o a n i l i n e (NA) (Remy e t al.,
1981) according t o the equation
B69
GPNA
The e f f l u e n t was monitored by t h e measurement o f the absorbance a t 410 nm. 8 . 2 . 3 Lactate dehydrogenase isoenzymes
The most i n t e n s i v e l y st udied isoenzyme f a m i l y are l a c t a t e dehydrogenases, owing t o t h e i r importance f o r diagnost ic purposes. Lactate dehydrogenase (LD) i s a tetramer o f Mr = 150 000, cont aining two s t r u c t u r a l l y d i s t i n c t subunits (M from s k e l e t a l muscle and H from h e a r t muscle). Various combinations o f these subunits l e a d t o f i v e d i f f e r e n t isoenzymes o f the same a c t i v i t y , LDHl
-
LDH5
H4, H3M, H2M2, HM3 and M4, r e s p e c t i v e l y ) . A t h i r d subunit, C, was found i n t e s t i c u l a r t i s s u e and spermatozoa. C u s u a l l y occurs as tetramer C4, b u t some (i.e.,
h y b r i d i z e d C+H+M isoenzymes were a l s o found i n nature (Evrev, 1975). The o x i d a t i o n o f l a c t a t e t o pyruvate using the oxidized form o f nicotinamide adenine di nucl eot ide, NAD',
i s t he p r i n c i p a l r e a c t i o n used i n a l l instances f o r
the post-column det ect ion o f these isoenzymes: C H~C H (OH )C OOH + NAD+
LD
C H ~ C O C O O H + NADH + H+
(*3407 E457) The reduced c o f a c t o r , NADH, can be cont inuously monitored e i t h e r by measurement
of the absorbance a t 340 nm o r by fluorescence ( e x c i t a t i o n a t 457 nm). The fluorescence method has t he g r e a t advantage o f higher s e n s i t i v i t y . More enzymec a t a l yse d reacti ons can be coupled t o t h i s redox reagent (cf.,
Section 8.2.4).
Kudirka e t a l . (1976) used f o r t he d e t e c t i o n o f LD a f t e r anion-exchange HPLC separation the above r eact ion, r e a l i z e d w i t h a Technicon AutoAnalyzer I1 s i n g l e channel col ori met er , where t he absorbance a t 340 nm was measured. Chang e t a l . (1976) used th e p r i n c i p l e i l l u s t r a t e d i n Fig. 5.29 (i.e.,
the supply o f the
s u bstrate through a union tee i n t o t he l i n e leading t o a packed r e a c t i o n column). I n contrast, Schroeder e t a l . (1977) described the p r i n c i p l e o f single-stream
'1 I "i
B
133 U 0
I2
LD2
L,
+
O! 0
I I
I
I
I I
TIME, MIN
I I
I
I
I I
I 32
Fig. 8.1. (A ) Chromatogram of serum LD isoenzymes from a patient with myocardial infarct. The upper trace was recorded a t the downstream detector (detector 2). The lower trace shows t h e background absorbance, a s i t was observed a t the upstream detector (detector 1 ) ( c f . , Fig. 5.31). The total serum LD a c t i v i t y was 502 U/l. (B) Profile of serum LD isoenzymes resulting from the correction f o r background absorbance (cf., A ) . The percentages of the total area a r e a s follows: LD5, 2.5; LD4, 1.2; LD3', 6.5; LD3, 4.4; LD2, 28.4; LDI, 56.9%; the high values of LDI and LD2 a r e significant f o r the diagnosis of infarction. (Reprinted from Ful t o n e t a l . , 1979.)
B71 o r p a r a l l e l -stream r e a c t i o n detectors based on an open-tubular system (an empty r e a c t i o n c o i l 1. Both absorbance and fluorescence monitoring were used. The postcolumn on-line detection o f LD isoenzymes was f u r t h e r i n v e s t i g a t e d and developed by Schlabach and co-workers (1977, 1978, 1980a,b) , Schlabach and Regnier ( 1 9 7 8 ) , Denton e t a l . (1979) and Fulton e t a l . (1979). L i v e r o r muscle damage, myocardial damage and some other accidents and pathological cases can be q u i c k l y detected by LO chromatographic p r o f i l i n g o f a small sample o f serum. Fig. 8.1 shows the r e s u l t o f the a p p l i c a t i o n o f a computer-controlled dual-detector post-column r e a c t i o n system t o LD serum p r o f i l i n g using background subtraction. D e t a i l s o f t h e various d e t e c t o r systems were discussed i n Section 5.2.3. LD chromatographic p r o f i l i n g w i t h o n - l i n e post-column d e t e c t i o n methods was reviewed by Regnier e t a l . ( 1 9 7 7 ) , Regnier and Gooding (1981) and Vacik and Toren ( 1982). 8.2.4
Hexokinase isoenzymes and 3-phosphogZycerate kinase
The post-column o n - l i n e detection o f hexokinase isoenzymes i s an example o f coupled enzyme assays. The enzyme monitoring methods can be c l a s s i f i e d i n t o two groups: (a) d i r e c t detection, where t h e immediate enzymic product i s monitored (Sections 8.2.1
- 8.2.3
g i v e several examples); (b) coupled enzyme assays,
where an a d d i t i o n a l enzyme o r more a d d i t i o n a l enzymes are required t o convert the product o f the primary enzyme r e a c t i o n i n t o a more e a s i l y detectable form. Schlabach and Regnier (1978) described the f i r s t method and a l s o the a p p l i c a t i o n o f the second method t o the detection o f hexokinase (HK) isoenzymes. They a l s o evaluated the e f f i c i e n c y o f both f r e e and immobilized coupling enzyme(s1. The measured hexokinase catalyses t h e phosphorylation o f D-glucose t o D-gl ucose-6-phosphate (G-6-P) using adenosine-5-triphosphate
(ATP) , and
adenosine-5-diphosphate (ADP) i s released. The G-6-P formed i s then o x i d i z e d by nicotinamide adenine dinucl e o t i d e (oxidized form NAD')
t o g l uconolactone-6-
phosphate; t h i s r e a c t i o n i s catalysed by a coupling enzyme, glucose-6-phosphate dehydrogenase (G-6-PDH). The reduced coenzyme NADH can be determined by known methods ( c f .
, Section
8.2.3),
i.e.
, by
absorbance measurement a t 340 nm or,
b e t t e r , by fluorescence measurement a t 457 nm. The sequence o f reactions i s as f o l 1ows : HK
D-Glucose + ATP -D-G1ucose-6-phosphate 0-G1 ucose-6-phosphate + NAD'
G-6-PDH
+ ADP
NADH + D-G1 uconolactone-6-phosphate + H+
(A340)
The coupling enzyme G-6-PDH must be added together w i t h the primary substrate D-glucose and other reagents (ATP + NAD')
t o t h e s o l u t i o n pumped i n t o t h e reactor.
However, the coupling enzyme i s o n l y a c a t a l y s t and i s n o t consumed i n t h e react i o n . It can be saved when used i n the r e a c t o r i n an immobilized form. This i s the general p r i n c i p l e of how t o save the a u x i l i a r y enzymes, which a r e sometimes expensive, i f they have t o be added t o the substrate s o l u t i o n i n a r e l a t i v e l y l a r g e amount and then f l o w from t h e r e a c t o r t o waste. Hexokinase isoenzymes were detected i n r a t l i v e r and t e s t i c u l a r t i s s u e e x t r a c t s using immobilized G-6-PDH i n a column reactor. For t h e detection o f HK, Lowe e t a l . (1981) used i n t h e i r "universal assay medium" t h e oxidized form o f nicotinamide adenine d i n u c l e o t i d e phosphate (NADP') instead o f NAD'
i n the above coupling reaction, and t h e reduced form NADPH was
obtained as the r e a c t i o n product, which absorbed a t 340 nm. For the detection of 3-phosphoglycerate kinase (PGK) a c t i v i t y , the f o l l o w i n g sequence o f reactions was used, where the coupling enzyme glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) was applied: 3-Phosphoglycerate + ATP
PGK
Glycerate-I ,3-biphosphate + ADP
Glycerate-l,3-biphosphate + NADH + H+
G-3-PDH
Glyceraldehyde-3-phosphate
t
(A340) t
NAD' + Phosphate
The "universal" assay medium contained both NADP' ( i n the oxidized) and NADH ( i n the reduced) form. I t i s evident t h a t the presence o f hexokinase i n t h e column e f f l u e n t produces an increase i n absorbance a t 340 nm, whereas 3-phosphoglycerate kinase a c t i v i t y decreases the absorbance a t 340 nm. The problems connected w i t h t h i s approach were discussed. 8.2.5
Creatine kinase isoenzymes
Creatine kinase (CK) i s a diiner o f Mr = 86 000 and i s composed o f M (muscle) o f B ( b r a i n ) subunits. Sometimes t h i s enzyme was designated c r e a t i n e phosphokinase, CPK. Three isoenzymes, CK, ( o r CPK,) t o CK3 (CPK3), can be derived from subunit combinations, i.e.,
BB, MB and MM, r e s p e c t i v e l y . CK i n b r a i n c o n s i s t s
o f BB o r contains B subunit, whereas MM predominates i n s k e l e t a l muscle and MB i s t y p i c a l of h e a r t muscle. CK isoenzyme p r o f i l i n g i s very important f o r the precise diagnosis o f myocardial i n f a r c t i o n , i n which MB isoenzyme i s elevated, i n c o n t r a s t t o the s i t u a t i o n i n coronary i n s u f f i c i e n c y .
CK c a t a l y s e s t h e phosphoryl a t i o n o f c r e a t i n e u s i n g adenosine-5-triphosphate (ATP), and t h e r e f o r e t h e systematic name o f t h i s enzyme i s ATP:creatine phosphotransferase. However, t h i s r e a c t i o n tends t o go i n t h e o p p o s i t e d i r e c t i o n : CK 0 Phosphocreatine + ADP I Creatine + ATP; AG = -3 kcal/mol The l i b e r a t e d ATP can be determined i n d i f f e r e n t ways. A method u s i n g a b i o luminiscence r e a c t i o n w i t h f i r e f l y l u c i f e r i n ( B o s t i c k e t a l . t i o n e d i n Section 6.6.4.
1980) was men-
Other methods employing c o u p l i n g enzymes w i l l be
described here. ATP can r e a c t w i t h 0-glucose using c a t a l y s i s by hexokinase (HK) and t h e D-glucose-&phosphate formed can be determined by o x i d a t i o n w i t h nicotinamide adenine d i n u c l e o t i d e (NAD')
(Sections 8.2.3
and 8.2.4),
or with
nicotinamide adenine d i n u c l e o t i d e phosphate m A D ( P ) + I :
ATP + D-glucose
HK -+
D-glucose-6-phosphate + ADP
0-G1 ucose-6-phosphate + NAD( P)'
G-6-PDH
D-gluconolactone-6-phosphate +
+ NAD(P)H+ H+ Schlabach e t a l . (1977) and Schlabach and Regnier (1978) described t h i s procedure. The use o f e i t h e r NAD' o r NAD(P)+ depends on whether t h e G-6-PDH c o u p l i n g enzyme i s from yeast o r from t h e microbe Leuconostoc mesenteroides. The l a t t e r p r e f e r s NAD',
whereas t h e y e a s t enzyme i s s p e c i f i c f o r NAD(P)+. The authors a l s o
t e s t e d and discussed t h e a p p l i c a t i o n o f immobilized c o u p l i n g enzymes. Denton e t a l . (1978, 1979) described t h e o n - l i n e m o n i t o r i n g of CK isoenzymes by use o f an immobilized enzyme microreactor. Other c o n t r i b u t i o n s t o t h e development o f CK post-column d e t e c t i o n were published by Schlabach e t a l . (1978, 1980a).
Interferences appearing i n t h e f l u o r i m e t r i c a l l y measured p r o f i l e s o f CK i s o enzymes i n human serum, due t o serum albumin, l i p o p r o t e i n and prealbumin, were discussed by Schlabach e t a l . (1980b). E l k i n s (1977) described an a l t e r n a t i v e method f o r t h e determination o f CK isoenzyme a c t i v i t y by t h e a p p l i c a t i o n o f an anion-exchange column and a c e n t r i f u g a l analyser. The HPLC o n - l i n e m o n i t o r i n g o f CK isoenzymes was discussed and reviewed by Regnier e t a l . (1977) , Regnier and Gooding (1981) and Vacik and Toren (1982).
874
8.2.6
ArylsuZphatase isoenzymes
Bostick e t a l . (1978) described t h e anion-exchange separation o f two a r y l sulphatase isoenzymes, A and B y i n human u r i n e using continuous d e t e c t i o n based on p-nitrocatechol sulphate hydrolysis. The product f i r s t passes through the f i r s t (reference) c e l l and, a f t e r a l k a l i n i z a t i o n w i t h NaOH, i t passes through the second (sample) c e l l , i n which t h e c o l o u r s p e c i f i c f o r t h e arylsulphatase product i s monitored. This enzyme a l s o hydrolyses p-nitrophenol sulphate, b u t the n a t u r a l substrate f o r arylsulphatase A i s cerebroside sulphate, which accumulates i n the body i n the case o f a deficiency o f t h i s enzyme. Bostick e t a l . (1978) a l s o described the separation and a n a l y s i s o f a r y l sulphatase isoenzymes i n o t h e r human body f l u i d s . Sera from p a t i e n t s w i t h c o l o r e c t a l cancer were examined. Arylsulphatase was a l s o mentioned i n a review by Regnier and Gooding (1981), describing the r a p i d separation o f p r o t e i n s i n c l u d i n g isoenzymes f o r the purpose o f c l i n i c a l analysis. 8.3 EXAMPLES OF ENZYME SEPARATIONS 8.3.1
Proteolytic enzymes
Buchholz e t a l . (1982) used SEC on LiChrosorb D i o l t o study changes i n enzymes i n s o l u t i o n , e.g., f o r the r a p i d a n a l y s i s o f t r y p s i n a u t o l y t i c degradat i o n . Fig. 8.2 i l l u s t r a t e s t h a t the decrease i n the height o f the f i r s t chromatographic peak f o l l o w s t h e decrease i n enzymic a c t i v i t y o f the enzyme s o l u t i o n , suggesting t h a t the f i r s t peak i n HPLC corresponds t o the i n t e g r a l a c t i v e enzyme. S t r i c k e r e t a l . (1981) p u r i f i e d m i l l i g r a m amounts o f commercially prepared bovine t r y p s i n (TRY) by RP-HPLC. T i t a n i e t a l . (1982) described a simple and r a p i d p u r i f i c a t i o n o f commercial TRY and chymotrypsin (CHY) by RP-HPLC, using aceton i t r i l e i n d i l u t e t r i f l u o r o a c e t i c a c i d a t pH 2. The enzymes were prepared i n amounts appropriate f o r the s t r u c t u r a l a n a l y s i s o f proteins. Each p u r i f i e d enzyme showed the s i n g l e expected substrate s p e c i f i c i t y . Strop and Cechovd (1981) separated a- and B-trypsin by H I C on both a n a l y t i c a l and preparative scales. Gooding e t a l . (1984) described the a n a l y s i s o f p r o t e i n s w i t h new, m i l d l y hydrophobic HPLC packing m a t e r i a l s (cf., Fig. 7.6). I n another p a r t o f t h i s work, the a c t i v i t y o f TRY was monitored using post-column o n - l i n e d e t e c t i o n w i t h benzoyl-D,L-arginine
p - n i t r o a n i l i d e (BANA) ( c f .
, Section
8.2.2).
875
I
I
I
50 100 Time (min)
I
150
F i g . 8.2. A u t o l y t i c degradation o f t r y p s i n i n s o l u t i o n as a f u n c t i o n o f time, f o l l o w e d by t h e decrease i n t h e h e i g h t o f t h e f i r s t peak i n t h e HPLC t r a c e ( 7 ) and by measurement o f t h e enzymic a c t i v i t y u s i n g t h e s u b s t r a t e N-a-benzoyla r g i n i n e p - n i t r o a n i l i d e (0). ( R e p r i n t e d f r o m Buchholz e t al., 1982.) Another group o f workers used a f f i n i t y chromatography f o r t h e r a p i d i s o l a t i o n o f p r o t e o l y t i c enzymes. Kasche e t a1 , (1981) immobil i z e d soybean t r y p s i n i n h i b i t o r as a b i o s p e c i f i c adsorbent by t h e g l u t a r d i a l d e h y d e method on aminos i l a n i z e d LiChrospher. a- and 6-TRY were separated w i t h a pH g r a d i e n t and chymotrypsinogen and a-CHY were separated a t c o n s t a n t pH. The k i n e t i c s o f t h e
, Section Works , Prague,
Turkovd e t a l . (1981) used Separon H
separation were s t u d i e d ( c f .
3.9).
(Laboratory Instrument
Czechoslovakia) d e r i v a t i z e d w i t h
e p i c h l o r o h y d r i n as a support f o r l a r g e - s c a l e HPLAC. F o r t h e p r e p a r a t i o n o f a s p e c i f i c sorbent f o r c a r b o x y l i c proteinases, t h e l i g a n d E-aminocaproyl-L-PheD-Phe-OCH3 was synthesized and c o v a l e n t l y attached t o t h e support (see a1 so Section 4.4.5).
The p r o t e i n a s e s from r a w pepsin and AspergiZZus oryzae were
i s o l a t e d u s i n g t h i s s o r p t i o n m a t e r i a l . S p e c i f i c sorbents f o r high-performance l i q u i d a f f i n i t y chromatography (HPLAC) and t h e l a r g e - s c a l e i s o l a t i o n o f proteinases were a l s o d e a l t w i t h by Turkovd (1982). Small e t a l . (1981) used packings c o n t a i n i n g immobilized t r i a z i n e dyes ( c f . HPLAC o f carboxypeptidase (CP) 6-2,
, Section
4.4.5)
f o r the
i n a d d i t i o n t o o t h e r enzymes and p r o t e i n s ;
f o r t h e d e t e c t i o n o f CP t h e change i n absorbance a t 320 nm was f o l l o w e d , when methotrexate (4-amin0-N~~-methylpteroylglutamate)was hydrolysed t o 2,4-diamino10 N -methylpteroate (McCullough e t al., 1971). Marceau e t a l . (1983) described t h e r a p i d assay o f human plasma carboxypeptidase N b y HPLC s e p a r a t i o n o f h i p p u r y l
B76 l y s i n e and i t s products. Wunderwald e t a l . (1983) studied t h e removal o f endoproteinases from b i o l o g i c a l f l u i d s by "sandwich a f f i n i t y chromatography" w i t h a2-macroglobulin bound t o zinc chelated Sepharose. TRY, CHY, thermolysin, elastase, bromelain, f i c i n and papain were bound, b u t n o t exoproteinases such as carboxypeptidases A and Y. The simple loading procedure, simple regeneration and h i g h capacity are advantages o f the method (see a l s o Section 7.4.3).
Shimura
e t a l . (1984) described the HPLAC o f plasmin and plasminogen on a h y d r o p h i l i c v i n y l polymer gel (Toyopearl HW 65 S ) w i t h p-aminobenzamidine. The column packed w i t h t h i s material r e t a i n e d both plasmin and plasminogen. Plasminogen was e l u t e d w i t h 6-aminohexanoic a c i d (a haptenic compound f o r t h e l y s i n e - b i n d i n g s i t e o f plasminogen). For the e l u t i o n o f plasmin, t h e coexistence o f 6-aminohexanoic a c i d and leupeptin (a competitive i n h i b i t o r o f plasmin) was necessary. Monitoring was e f f e c t e d by f l u o r i m e t r i c d e t e c t i o n o f the e l u t e d p r o t e i n and one-line assay o f plasmin a c t i v i t y using peptidylmethylcoumarylamide, a fluorogenic substrate (cf.,
Fig. 5.18).
Gooding and Schmuck (1983) p u r i f i e d TRY and o t h e r basic p r o t e i n s (CHY, lysozyme and cytochrome c) by cation-exchange HPLC. A SynChropak CM 300 column was used and sodium acetate proved b e s t f o r i o n i c strength gradient e l u t i o n , TRY a c t i v i t y was monitored using BANA (see Section 8.2.2) reagent GPNA was used (see Section 8.2.2).
and f o r the d e t e c t i o n o f CHY the Cohen e t a l . (1984) demonstrated
m u l t i p l e peak formation i n the RP-HPLC o f papain: t h e f i r s t peak was n a t i v e and the second was p a r t o f the enzyme, denatured i n contact w i t h t h e column. 8.3.2
C e l Z u l o l y t i c , p e c t o l y t i c and amyZoZytic enzymes
Montenecourt e t a l . (1980) studied the biochemical nature o f c e l l u l a s e s from t w o physical hypercel l u l o l y t i c mutants o f Trichoderma r e e s e i using HPLC on DEAEs i l i c a (Applied Science Labs.,
State College, PA, U.S.A.).
Buchholz e t a l . (1982) c o r r e l a t e d the a c t i v i t y and the molecular s i z e o f c e l l u l a s e components using high-performance SEC on LiChrosorb Diol columns. For t h e determination o f the c e l l u l a s e a c t i v i t i e s , glucanases were analysed by incubation w i t h Avicel; samples were taken a t i n t e r v a l s , c e n t r i f u g e d and analysed f o r glucose and c e l l o b i o s e by HPLC on a LiChrosorb-NH2 (Knauer) column w i t h a c e t o n i t r i l e - w a t e r as t h e eluent and a d i f f e r e n t i a l refractometer detector. a-Glucosidases were determined by incubation w i t h c e l l o b i o s e and analysis as before. The main a c t i v i t i e s were found i n the main peaks. Hostomskd and Mikes (1983) described the a n a l y t i c a l MPLC o f c e l l u l o l y t i c enzymes on Spheron ion-exchangers and a l s o (Hostomski and Mikes, 1984) separated the c e l l u l o l y t i c system o f Trichoderma u i r i d e - r e e s e i mutant by MPLC on the l a r g e preparative scale and i s o l a t e d a new ezo-cellobiohydro1ase.
B77 MikeS e t a l . (1981) s t u d i e d t h e MPLC s e p a r a t i o n o f p e c t i c enzymes (endo-Dgalacturonase, exo-D-galacturonase,
p e c t i n l y a s e and p e c t i n esterase) from
Pectinex U1 t r a ( o r i g i n a t i n g f r o m A s p e r g i l l u s niger f e r m e n t a t i o n ) and Rohament P t e c h n i c a l p e c t o l y t i c p r e p a r a t i o n s . A l l a v a i l a b l e Spheron i o n exchangers were t e s t e d f o r t h e separation o f these p e c t i c enzymes. The experience f r o m these s t u d i e s was used f o r a s i m i l a r a n a l y s i s o f p e c t i c enzymes (Rexovd-Benkovd e t al., 1982) i n t h e Czechoslovak t e c h n i c a l p e c t o l y t i c p r e p a r a t i o n Leozym, which i s a by-product i n t h e manufacture o f c i t r i c a c i d by f e r m e n t a t i o n o f A s p e r g i l l u s n i g e r . MikeS and Rexovd (1988) reviewed techniques f o r t h e HPLC o f p e c t i c en-
zymes. Fourmy e t a l . (1982) described a r a p i d q u a n t i t a t i v e method f o r t h e d e t e c t i o n o f macroamylase i n human serum by HPLC (SEC). Isoamylases f r o m s a l i v a and p a n c r e a t i c j u i c e were a l s o analysed. Serum f r o m normal persons c o n t a i n e d two amylase peaks, d i s t a n t from t h e v o i d volume. The second peak was markedly h i g h e r i n serum from p a t i e n t s w i t h acute p a n c r e a t i t i s and mumps. I n c o n t r a s t , t h e amylase a c t i v i t y i n serum from p a t i e n t s w i t h macroamylasaemia was e l u t e d i n t h e v o i d volume. 8.3.3
Oxidoreductases
High-performance 1 i q u i d a f f i n i t y chromatography (HPLAC) has been used succ e s s f u l l y f o r t h e separation o f many enzymes. Horse 1 i v e r a l c o h o l dehydrogenase (LADH) and p i g h e a r t l a c t a t e dehydrogenase (LDH) were w e l l separated from each 6 o t h e r and f r o m bovine serum albumin on an N -(6-aminohexyl)-AMP bonded s i l i c a column. B i o s p e c i f i c e l u t i o n u s i n g d i l u t e s o l u t i o n s o f NAD+-pyrazole and NAD+pyruvate were used, i n a d d i t i o n t o a h i g h c o n c e n t r a t i o n o f sodium c h l o r i d e s o l u t i o n . When a g r a d i e n t o f NADH was applied, and M4 LDH isoenzymes (Ohlson e t a l . ,
i t was p o s s i b l e t o separate H4
1978). Lowe e t a l . (1981) used Cibacron
Blue F3G-A bonded s i l i c a f o r t h e f u l l y automated HPLAC r e s o l u t i o n o f dehydrogenases (such as LADH and LDH) , i n a d d i t i o n t o hexokinase, 3-phosphoglycerate kinase and o t h e r enzymes. Simultaneous d e t e c t i o n was achieved by m o n i t o r i n g t h e change i n absorbance a t 340 nm ( c f . ,
Section 8.2.4).
Small e t a l . (1981)
immobilized a number o f t r i a z i n e dyes t o m i c r o p a r t i c u l a t e s i l i c a and s t u d i e d t h e i r a p p l i c a t i o n f o r t h e s e p a r a t i o n o f LDH and o t h e r enzymes. B o r c h e r t e t a1
.
(1982) immobilized concanavalin A t o porous s i l i c a and used t h i s support f o r t h e a n a l y s i s and p u r i f i c a t i o n o f glucose oxidase and g l u c o p r o t e i n s peroxidase. This HPLAC method was s t u d i e d i n d e t a i l . I E X has a l s o o f t e n been used f o r t h e r a p i d s e p a r a t i o n o f enzymes. Matsumoro (1981) described t h e t h e o r y and use o f serum LDH isoenzyme a n a l y s i s on I E X 525
878
QAE; w i t h a gradient of NaCl concentration t h e t o t a l separation o f isoenzymes was achieved w i t h i n 20 min. Van der Wal and Huber (1980) studied t h e performance of c l a s s i c a l n o n - r i g i d ion-exchange packings o f small p a r t i c l e s i z e i n the separation of cytochrome c and d e r i v a t i v e s by HPLC, b u t b e t t e r r e s u l t s were obtained w i t h (meth)acryl i c c a t i o n exchangers and hydroxyapatite. Vanecek and Regnier (1982) described a r a p i d separation o f lipoxygenase I using a column o f LiChrospher S i 4000 coated w i t h a heavy l a y e r o f polyethylenimine and crossl i n k e d w i t h 1,4-butanediol
d i g l y c i d y l ether. Lindblom (1983) developed a simple
method f o r the i s o l a t i o n o f glucose-6-phosphate dehydrogenase from a yeast enzyme concentrate. A column o f Polyanion S I (8 pm) was used f o r a n a l y t i c a l and Polyanion S I (17 pm) f o r preparative purposes. Hearn e t a l . (1980) studied the SEC o f sheep l i v e r aldehyde dehydrogenase (and t h y r o g l o b u l i n ) on pBondage1 E-linear. I n d i c a t i o n s o f the i n f l u e n c e o f a mixed-mode separation p r i n c i p l e were found. Power e t a l . (1983) used RP-HPLC f o r the p u r i f i c a t i o n o f nuclear coded subunits o f a membrane oligomeric p r o t e i n
-
yeast cytochrome c oxidase. 8.3.4 Enzymes of phosphate metaboZism, other miscellaneous enzymes and enzymic
reactions Small e t a l . (1981) used dye-ligand chromatography f o r the f r a c t i o n a t i o n o f yeast hexokinase on Procion Green H-4G s i l i c a , and of L-tryptophanyl - t R N A synthetase o f Cibacron Blue F3G-A s i l i c a and Procion Brown MX-5BR s i l i c a . Lowe e t a l . (1981) studied chromatography on Cibacron Blue FSG-A s i l i c a and detection methods f o r hexokinase, 3-phosphoglycerate kinase ( c f . , Section 8.2.4)
and a l s o
o f pancreatic ribonuclease A. Mikes e t a l . (1978) described t h e r a p i d I E C separation o f technical enzymes k.g.,
crude protease from AspergiZZus sojae and glucose oxidase from Aspergiltus
niger (Fig. 8.3)]. Kato e t a l . (1980) p u r i f i e d crude 8-galactosidase from b a c t e r i a l c e l l s and commercial urease by SEC on TSK Gel 3000 SWG. Roughly 15-fold p u r i f i c a t i o n was achieved i n a s i n g l e f i l t r a t i o n . Lim (1979) developed HPLC methods f o r the determination o f enzymes o f the haeme b i o s y n t h e t i c pathway (6-aminolaevul i n i c a c i d synthetase and dehydratase, uroporphyrinogen I synthetase). The enzymes were n o t isolated, b u t determined by the separation of t h e i r low-molecular-weight products. Studebaker (1979) reviewed the analysis o f enzymic reactions by HPLC.
879
25
a
0 I
I
10
20
I
30
F.N.
Fig. 8.3. Chromatography of technical glucose oxidase on a DEAE-Spheron column (20 crn x 0.8 cm). Load: 15 mg of preparation in 0.2 m l of buffer A. The ion exchanger was e q u i l i b r a t e d w i t h b u f f e r A. I , Buffer A without g r a d i e n t . Linear gradients I1 ( A + B ) and I11 ( B + C). IV, buffer C without g r a d i e n t . Flow-rate, 2 ml/min; 4-ml f r a c t i o n s ; temperature, 14oC; counter pressure, 3-7 atm; c h a r t speed, 2 mm/min. Buffers: A, 0.01 M a c e t i c a c i d + NaOH, pH 6.8; B y 0.3 M a c e t i c acid + NaOH, pH 5.5; C , b u f f e r B y 1 M in NaC1, pH 5.3. Broken l i n e , glucose oxidase a c t i v i t y of e f f l u e n t i n S a r r e t u n i t s (S.U.; 1 S.U. corresponds t o t h e consumption of 600 p1 of oxygen a t 30oC). F.N. = Fraction number; M = automatic marking of f r a c t i o n c o l l e c t i o n ; mS = e l e c t r i c conductivity in mS. Corresponding peaks in both p a r t s o f t h e chromatogram a r e designated by a - i . The UV spectrum of the chromophore o f compound d d i f f e r e d s i g n i f i c a n t l y from t h e others. (Reprinted from Mike: e t a l . , 1978.) 8.4 COMMENTS ON LITERATURE G u i l b a u l t ' s (1976) Handbook i s not devoted t o HPLC methods, b u t i t describes in d e t a i l assay procedures f o r 39 important enzymes, enzymic analyses of more than 55 s u b s t r a t e s and t h e a p p l i c a t i o n of immobilized enzymes in enzymic analyses, and provides much additional p r a c t i c a l information. Regnier e t a l . (1979) and Regnier and Gooding (1981) reviewed t h e HPLC of p r o t e i n s , including t h e HPLC of enzymes; these reviews cover specialized a p p l i c a t i o n s in c l i n i c a l a n a l y s i s . Ishiguro and Shinohara (1981) reviewed t h e use of IEC, SEC, a f f i n i t y , adsorption and high-performance 1 iquid chromatography in the separation o f isoenzymes (especially of l a c t a t e dehydragenase, c r e a t i n e phosphate kinase, glutamate-
B80
o x a l a c e t a t e transaminase, a1 k a l i n e phosphatase and ma1 i c dehydrogenase). Mikes
(1981/1982 and 1982) d e a l t w i t h t h e r a p i d chromatographic a n a l y s i s o f enzymes and o t h e r p r o t e i n s f o r a p p l i c a t i o n t o f o o d c h e m i s t r y . Vacik and Toren (1982) reviewed t h e s e p a r a t i o n and measurement o f isoenzymes and o t h e r p r o t e i n s by HPLC; t h i s t r e a t i s e covered p r e d o m i n a n t l y b i o m e d i c a l a p p l i c a t i o n s . Enzyme s e p a r a t i o n by RP-HPLC was reviewed by S t r i c k l e r e t a l . (1984). Jakoby
(1984) e d i t e d a s p e c i a l volume o f Methods in EnzymoZogy d e a l i n g w i t h t h e p u r i f i c a t i o n o f enzymes, where HPLC t e c h n i q u e s f o r enzyme s e p a r a t i o n s were d e s c r i b e d i n d e t a i l ; Wehr d e a l t w i t h t h e c a r e o f HPLC columns, Unger e x p l a i n e d SEC methods, Regnier I E C , Richey o p t i m a l pH c o n d i t i o n s f o r I E C , Hearn RPC and Larsson HPLAC.
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