red n z
Howard H. Weetall Corning Glass Works Corning. N . Y 14830
Enzymes can be immobilized or confined on or in a variety of watersoluble and water-insoluble matrices with little or no immediate loss of their catalytic activity. This is of interest and importance for theoretical reasons. Most enzymes are not found randomly dissolved in the cytosol or cytoplasm but are attached to membranes in or on various cell organelles. Further, the physicochemical characteristics of these “insolubilized” enzymes are affected by the microenvironments in which they are found. When enzymes are removed from their microenvironments, their behavior may differ. Immobilization of soluble enzymes on or in synthetic membranes or on organic or inorganic matrices permits researchers to study them in environments modeled after their natural states. A second and more practical important reason for immobilizing enzymes is that immobilized enzymes allow researchers to add, remove, and reuse enzymes a t will. This also means t h a t industrial processors presently using soluble enzymes may, in some cases, economically substitute immobilized enzyme systems. Similarly, the ability to remove and reuse these materials makes them likely 602A
candidates for use in the analytical and clinical laboratory and even in the physician’s office. Kot a new technology, enzyme immobilization can be traced back a t least 50 years to the work of Nelson and Griffin ( I ) . These workers adsorbed invertase to animal charcoal and observed that the “immobilized enzyme” retained biological activity and could be reused over a long period of time. For the most part, these studies went unnoticed until the 1950’s when Grubhofer and Schlecth immobilized several enzymes on polyaminopolystyrene and on a chlorinated resin Amberlite XE-64 by covalent attachment (2. 3). It was around this time that other methods of enzyme immobilization atso came into prominence. Methods of Enzyme Immobilization There are several methods of immobilizing enzymes. [For a general review of immobilization, see Zaborsky ( 4 . 1 Absorption. Absorption of an enzyme to a water-insoluble surface is the simplest method of immobilization. The subject has been reviewed in depth by McLaren and Packer ( 5 ) . Adsorption. The adsorption of enzymes onto surfaces is dependent on many variables including pH, type of solvent, ionic strength, temperature. and enzyme concentration (6).The stability of the adsorbed enzyme and its activity are dependent on assay and storage conditions, substrate con-
A N A L Y T I C A L CHEMISTRY. V O L . 46, NO. 7. J U N E 1974
eS:
centration, ionic strength of storage solution and many of the same parameters which effect the adsorption phenomenon itself. Commonly used adsorbents include: alumina, carbon, celluloses, clays, hydroxyapatite, and glasses including controlled-pore glass. Messing has shown t h a t enzymes adsorbed to glass surfaces have extremely long half-lives and can be successfully used repeatedly without activity losses (7, 8).
Adsorption and Cross-Linking. Enzymes have been adsorbed to colloidal silica followed by cross-linking with glutaraldehyde (9). These enzymes retained their activity and could be reused several times. In a similar fashion Gaffield et al. and Olson and Stanley adsorbed lactase to a phenolformaldehyde resin and crosslinked the adsorbed enzyme with glutaraldehyde (20, 11).The technique is generally rapid and simple and produces a product of relatively good stability. Cross-Linking. Enzymes can be immobilized by cross-linking with low-molecular-weight multifunctional reagents producing covalent bonds with intermolecular cross-links between the reagent and the enzyme. The activity of the cross-linked enzyme is dependent on many factors including: concentration of enzyme, reagent, pH, ionic strength, and number of cross-links produced. The overall apparent activity of the derivative is also dependent on the size of the substrate. Generally, high-molecular-weight substrates cannot come in
Report
Analytical Applications
contact with enzymes in the interstices of such immobilized enzyme supports. Multifunctional agents most commonly used include diazobenzidine and its derivatives and glutaraldehyde. Other less-used agents include carbodiimides. diisothiocyanates, diisocyanates, and disulfonic acids. Ion-Exchange Resins. Enzymes can be immobilized through ion-exchange techniques. Many enzymes will bond to ion-exchange resins without significant loss of activity. Unless the pH or ionic conditions are changed to cause elution of the enzyme. it will remain attached and active. This approach has successfully been used in immobilizing L-aminoacylase on DEAE-Sephadex. This immobilized enzyme is used commercially for production of L-methionine at a rate greater than 20 metric tons per month. E n t r a p m e n t . Enzymes can be immobilized by entrapment within cross-linked water-insoluble polymers. The method of preparation usually involves the cross-linking of the polymer in the presence of the enzyme. physically entrapping the enzyme. The polymer lattice structure is such that the large enzyme molecule cannot diffuse out, but small substrate molecules can diffuse into the polymer. Gel entrapment was first successfully used by Bernfeld and Wan (12).Materials used for entrapment include: polyacrylamides. silicone rubber, silica gel. and starch. Microencapsulation. Enzymes have been immobilized by encapsula-
tion within semipermeable membranes. The encapsulation is carried out by an interfacial polymerization or coacervation of a preformed polymer. The first reports of encapsulating enzymes in permanent membranes were those of Chang (13).Enzymes have been encapsulated in membranes of collodian. polystyrene, and cellulose derivatives ( 4 ) . The most common polymer used for encapsulation, however, is nylon. Copolymerization. Enzymes can become immobilized by being covalently incorporated into polymers. The most common materials for copolymerization are maleic anhydride and ethblene, which were first reported by Bar-Eli and Katchalski (14). Copolymerized enzymes can be prepared as either neutral or charged derivatives. As with entrapped and microencapsulated enzymes, these derivatives show little or no actibity with large macromolecular substrates. Covalent Attachment. Chemical attachment of enzymes to water-insoluble carriers is the most commonly used method of immobilization. Theoretically, covalent coupling offers the most stable, most versatile method of immobilizing enzymes. Methods of covalent attachment are too numerous to be discussed in a review of this nature. However, some of the more common methods of attachment and carriers are as follows: Carbox? rneth? lcellulost~A z i d e The carboxymethylcellulose is converted by the reactions schematically shown in Figure 1.The method is relatively
simple. The final coupling step is carried out a t a slightly alkaline pH. The final product is an amide formed primarily to the [-amino group of lysine. Other possible attachment sites include cysteine, serine, and tyrosine. Azo Linkage. Arylamines can be diazotized and coupled via azo linkage to proteins. The technique has been successfully used for covalent attachment of proteins to polyaminopolystyrene, p-aminobenzoylcellulose, and arylamine-glass (15).The coupling to protein occurs a t a slightly alkaline pH. The reaction is schematically represented in Figure 2 . Coupling occurs through an available tyrosine, although lysine, arginine, cysteine, and serine have been implicated in the reaction.
Zsocyana tes and Isothiocyanates. Arylamines and alkylamines can be converted to isocyanates and isothiocyanates. These activated derivatives will then react with amines on the protein, primarily the {-amino group of lysine. to form substituted ureas. The reactions are schematically represented in Figure 3. The coupling is carried out a t slightly alkaline pH. Carbodiirnides. Water-soluble carbodiimides can be used for covalently attaching proteins to derivatives through amide linkage. The enzyme may be coupled under slightly acid conditions. The reaction is schematically represented in Figure 4. Amide derivatives can also be used for coupling to the carboxyl group of the protein with carbodiimides, although under these conditions, cross-linking is quite possible.
A N A L Y T I C A L CHEMISTRY. VOL. 46, N O . 7 . J U N E 1974
603A
Figure 1. Preparation of carboxymethylcellulose azide and attachment of enzyme to active derivative
Figure 2. Preparation of diazonium chloride and attachment of enzyme through azo linkage
0 II
clccl
1).
[ R ~ N C O ISOCYANATE
\ R - @ W ? ~
C d CI 2). I R a N C O + "2-ENZYME
Figure 3. Preparation of isocyanate and isothiocyanate derivatives and their attachment to an enzyme
3).
R
~
N
-/
RaNCS +NHZ-ENzyw
R' I N
R' I NH I
N I R"
I R"
CISTHIOCYANATE S
0 RGNgNH-ENZYME S
R a N g "
-ENZYME
R' I
2).
Figure 4. Preparation of an active pseudourea from a carboxyl and a carbodiimide and reaction with an enzyme forming amide linkage
1
NH I
coot
+ ",-ENZYME
LH' I R"
-
NHR' I CONH-PROTEIN + o s C +H+ I NHR'
Figure 5. Preparation of cyanogen bromide-activated derivative and its attachment to an enzyme
604A
A N A L Y T I C A L C H M I S T R Y , VOL
46, N O 7 J U N E 1974
Your suggestions take form in Thomas Laboratory Specialties.
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C?anogen Hionid,r. T h e activation of cross-linked dextrans. iincluding agarose and even cellulose. is a simple and attractive method of covalently coupling preteins t o waterinsoluble carriers. The reaction sequence is schematically represented in Figure 5. The coupling !reaction works best a t pH 9.0, although many workers prefer a mow neutral pH. The t -amino lysine group is the group through which coup1 ing generally occurs to the protein. Giutaraide~i7.vde.The covalent a t tachment of protein: to water-insoluble carriers via glutaraldehyde is one of'the simplest and most gentle of coupling methods. The reaction is carried ciut at neutral pH. Attachment is trom the am:ne carrier to the t -amino group of'lysine in the protein. The reaction schematically represented in Figure 6 IS oversimplified for purposes of'this review. There are many other method; of covalently coupling enzymes to water-insoluble carriers. [For further details. see one of F.evera1available reviews (4. 16. 17, 1Hi.1
Characteristics of Immobilized Enzymes \+'hen a n enzyme is immobilizedeither u-ithin a matrix or on the surface o f a carrier--set.eral changes may occur in the enzyme s apparent behavior. The factors (Effecting this behavior are many. Hcwever. several of the observed changes occur quite commonly and will be considered here. pH Profile. All ei:zymes have a n optimal pH at which they show a maximum reaction rate. 'When the enzyme is immobilixed. the optimal pH may shift. depending on the nature of' the carrier. G oldst ein and coworkers (19)studied this phenomenon in detail. In a nutshell. they report: If a carrier is negatively charged. then a high concentration of positively charged ions (H' ) will accumulate at the boundary layer between the carrier and the surrounding solution. This accurnulation of hydrogen ions will cause the pH a t the carrier surface to drop below that of the bulk solution. The enzyme. therefore. sew a pH below that of the bulk solut i m . In this manner. the apparent pH ofthe immobilized enzyme may be increased. If the carrier ii, negatively charged. the opposire may occur (Figure 7 ) . Kinetics. For the most part. the kinetics of immobilized enzyme reactions are studied in term!; of "apparent" values. Severa 1 excellent discussions of'the kinetics of immobilized enzymes are available ( 1 6 . 20, 2 1 ) . When an enzvme is immobilized. one
n
U
> a: W
v,
m
0
LL
0 I-
Figure 7. p H profile of yeast lactase in solution and covalently attached to ZrOP-coated control pore glass, 550 A pore diameter, 4 0 / 8 0 mesh. Substrate was 10% lactose solution. (From ref. 34 i
z
W 0
a: W
0.
0 2.0
3.0
5.0 PH
4.0
6.0
7.0
Table I. Comparison of K, Values of Some Soluble and Immobilized Enzymes a K,, mM Enzyme
Invertase Arylsulfatase Glucoamylase Alkaline phosphatase Urease Glucose oxidase !--Amino acid oxidase
Substrate
Soluble
Immobilized
Sucrose p-Nitrophenylsulfate Starch p-Nitrophenylphosphate Urea Glucose
0.448 1.85
0.448 1.57
1.22 0.10
0.30 2.90
10.0 7.70
7.60 6.80
1.00
4.00
L-Leucine
All enzymes were immobilized on ZrOL-coated control porous 96% silica glass p a r ticles. K,, values were determined under identical conditions for both soluble and immobilized derivatives for comparison.
Table II. Comparison of Activation Energies of Some Soluble and lrnmobitized Enzymesa Activation energy, kcal/gmol Enzyme
Soluble
Immobilized
Papain (amide linkage) Papain (azo linkage) Glucose oxidase (azo linkage) Glucoamylase (Shiffs base) Yeast lactase (Shiffs base) Microbial lactase (Shrffs base)
13.8
11.0 13.8 9.0 13.8 11.3 6 5
...
6.6 16.3 10.5 10.4
Enzymes immobilized on inorganic supports. Comparative values were obtained between the same temperature ranges where possible.
ANALYTICAL CHEMISTRY
VOL
46. N O
7
J U N E 1974
6807A
Enzyme electrodes of many types and varieties are possible. For example. one can determine L-amino acid content with an ammonia-sensitive electrode using L-aminoacidoxidase. which deaminates L-amino acid as follows:
60 !=
z
SOLUBLE PAPAIN
40L
\
+
-
+ II 0 + 0 RCOCOO- + S H + H 0A
@\
20
TEMPERATURE ("C) in some cases increased by immobilization. T h i s figure compares papain in solution and immobilized on control pore glass to storage at increasing temperatures for 30-min intervals. Soluble enzyme was dissolved while immobilized derivative was suspended in water. Residual activity was measured by assay with 1% casein at 25°C (From ref. 32. Part I )
Figure 8. Thermal stability of an enzyme is
generally observes a n increase in
K,,, , This increase is usually related to the charge on the substrate and/or carrier, diffusion effects, and in some cases. tertiary changes in enzyme configuration. However. in some cases. no change in K,,, is observed (Table I ) . S t n h i i i t ? l i k e all other proteins. enzymes are susceptible to thermal denaturation. whether they are immobilized or in the "free" state. In many cases. however, the rate of inactivation and denaturation of a n immobilized enzyme is less than that of the free enzyme. The thermal stability of the enzyme papain is shown in Figure 8. Inactivation occurred a t lower temperatures with the free enzyme. Similar results have been observed with many other enzymes. Table I1 gives the activation energies for several soluble and immobilized enzymes;.Enzymes which show excellent thermal stability do not necessarily show excellent operational stability. because the operational stability ofimmohilized enzymes is not only a function of thermal stability. but o f such factors as carrier durabilit y and organic inhibitors and inhibitor concentrations, including that ofheavy metals. The clogging ofthe carrier also effects half-life-that point where the amount of activity of the enzyme is 50% of what it was when initially used. Enzyme half-lives under controlled operating conditions should be temperature-dependent. but this does not mean that the activity decrease is denaturation dependent. Table 111 gives a n example of the dependency of half-life on operating temperature for a n immobilized enzvme. 608A
RCHCOOTH
ANALYTICAL CHEMISTRY. VOL
Applications of Immobilized Enzymes Analytical Applications-Enzyme Electrodes. An enzyme electrode is basically a combination of an enzyme and a n electronic sensing device. Such a n electrode possesses the specific properties of the enzyme and the adaptability and ease of readout of a clinical electrode device. The first enzyme electrode as developed by Updike and Hicks (22) was prepared by entrapping the enzyme glucose oxidase in a gelatinous polymer coating over the surface of a polarographic oxygen electrode. When the electrode is placed in contact with a glucose solution, glucose and 0 2 diffuse into the gelatinous layer around the electrode where the diffusing glucose is oxidized. producing gluconic acid. The resulting depletion of oxygen ( 0 2 ) is measured by the oxygen electrode. At glucose concentrations below K , for the entrapped enzyme. Updike and Hicks found a linear relationship between glucose concentration and the measured 0 2 depletion rate. Guilbault (23)prepared a urea electrode by entrapping urease in a polyacrylamide matrix which is then placed over a cation-selective electrode. The urease hydrolyzes urea to ammonia and carbon dioxide. The rate at which ammonia is produced from the urea diffusing into the membrane is proportional to the quantity of urea present when operated at urea concentrations between 1.0 and 30.0 mg of urea per 100 ml of solution. Guilbault has shown that these electrodes are quite stable and can be operated continuously for at least three weeks. 46, NO
7 . JUNE 1974
The HZOZ can he removed by the addition of catalase. Similarly. specific amino acids can be quantitated with specific amino acid deaminases. Rather than the oxygen and cation-specific electrode. Clark used polarographic oxidation of H202 to measure glucose and amino acids ( 2 1 ) .He used a platinum electrode to which a n applied voltage causes oxidation or reduction and results in producing a current. The platinum electrode is operated at a voltage so that the current produced is proportional to H202 concentration. Clark identified some 30 enzymes which use 0 2 and produce H202. IJsing these enzymes with polarographic platinum electrodes. Clark estimated that the following compounds could he detected: .4cetaldehyde. D-mannose D -alan i ne. met han I 1 Aliphatic nitro compounds. L-methionine D-aspartate. 6-methyl-D-glucose Benzaldehyde. S-methyl-L-amino acids Diamines. S A D 2-Dioxy-D-glucose. NADH Ethanol. oxalate Formaldehyde. L-phenylalanine L -gala c t on01act one, D - pr 01 ine D-galactose. purine 3 -D-glucose. pyridoxamine phosphate D-glutamate. pyruvate Glycollate. saccosine L-gulono-h-lactone. spermine Hypoxanthine. sulfite D-lactate. tyramine L-lactate. urate Lactose. D-valine ( $ 1 Mandalate. xanthine Table 111. Half-Life and Temperature Vs. Productivity for Immobilized Glucoamylase Covalently Coupfed to ZrO&oated Porous Glass" Temp, O C
Half-life, days
Relative reaction rate, '% of that at 60%
60
13
50 45
100
100 70
645
30
40
900
25
"When plotted a s half-life vs. 1 / T (OK), a positive slope is observed. From the slope the deactivation can be calculated (53).
Nine reasons why the Varian 2740 is your best buy in a research GC I
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2
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Separate injector and detector temperature controls are continuously variable from ambient to 4OOOC allowing more precise adjustment for optimum performance. The 2740 includes every standard feature expected in a research G C plus many Varian extras that help you improve your analyses.
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A N A L Y T I C A L CHEMISTRY, VOL. 46, N O . 7, J U N E 1974
609A
Though not all the above compounds are of interest to the clinical and analytical chemist, the list indicates the wide range of compounds capable of detection with particular types of enzyme electrodes. Similarly, one can mass a list of compounds that produce or utilize oxygen or ammonia and are therefore quantitatable with a n oxygen or cation electrode. A third type of electrode system uses enzymes to quantitate either a substrate or a product. Developed by Baum and Ward (25),this is known as a n organic liquid ion-exchange electrode. Baum’s electrode specifically detected acetylcholine and thus could be used for the detection and quantitation of organophosphates which inhibit the activity of acetylcholinesterase. The reaction between the enzyme and product could be monitored by using the acetylcholinespecific electrode to determine the substrate depletion rate. Since the electrode could respond to a variety of choline compounds (Figure 9),it was useful with a variety of cholinesterase substrates. The electrode was used to perform studies on cholinesterase activity in blood. Results compared favorably with data obtained with established colorimetric and pH-stat techniques. Similarly, this electrode was successfully used to measure the presence of the organophosphate pesticides Paraoxon and Tetram by cholinesterase inhibition. Inhibitor concentrations of 10-100 ng/ml were quantitatable by this method. Baum later combined his cholinesensitive electrode with immobilized cholinesterase. The immobilized enzyme was in the form of a disc which was simply added to the solution to be quantitated. Immobilized cholinesterase discs were used extensively over a 64-day period with excellent results . Immobilized enzymes have also been used successfully for analytical purposes in the form of columns. Weibel et al. (27) developed a glucose analyzer that uses a column of immobilized glucose oxidase. As illustrated in Figure 10, besides the column of immobilized glucose oxidase, the system includes a Clark electrode to detect 0 2 depletion. and a pump. Though the system operates in either a kinetic or end-point mode, the endpoint mode is preferred. Weibel e t al. found that complete conversion of glucose could be accomplished in less than 60 sec with immobilized glucose oxidase columns containing only 400-600~1of porous glass. Using glutaraldehyde, Inman and Hornby covalently attached urease and glucose oxidase to partially hydrolyzed nylon power and tubes (28). 610A
ANALYTICAL CHEMISTRY, VOL
Figure 9. Selectivity of liquid organic ion-exchange electrodes toward choline and choline esters. (From ref. 2 5 )
Figure 10. Schematic representation of glucose monitor showing principal components. This monitor uses imrnobolized glucose oxidase in a column as opposed to a membrane electrode. (From ref. 27)
These derivatives have been successfully used in the automated analyses of glucose and urea. Glucose is determined spectrophotometrically as a function of the quantity of I2 produced in reacting KI with H202. T h e urea is determined by ammonia formation according to the method of Chaney and Marbach (29).The flow system, using nylon tubes, is illustrated in Figure 11. The system can process 60 samples per hour without difficulty. The stability of the tubes was determined by performing 150 assays per day for 30 days. During 46. N O
7, JUNE 1974
this period, no activity losses were noted. Similarly, the urease derivatives through 5000 assays showed no detectable loss in activity. The use of immobilized enzymes in automated systems is under intensive investigation in Hornby’s laboratory, where researchers are attempting to develop assays for a wide variety of clinically important substrates. Immobilized enzymes can also be used for detection and quantitation of inorganic ions. This author has developed an immobilized enzyme system for detecting and quantitating inor-
P
MCI
NYLON -TUBE -SUPPORTED ENZYME
.
I
Y
1
I
I
MC3
Figure 11. Flow diagram for use of nylon tube-supported enzyme automated system. For determination of glucose, lines 1-6 operate at predetermined flow rates. MC,mixing coil; HB, heating bath; S, samples; P, pump; SPEC, spectrophotometer; DB, debubbler; W, waste. (From ref. 28)
F R ACTtON COLLECTION
Figure 12. Schematic representation of apparatus for continuous monitoring of inorganic phosphate in solution (From ref. 30)
ganic phosphate and sulfate with immobilized alkaline phosphatase (30) and immobilized arylsulfatase, respectively. The reactions involved are: p -nitrophenolphosphate + H 2 0 (colorless) p -nitrophenol HP04 (yellow) +
+
p -nitrophenylsulfate
+
H,O
(colorless p -nitrophenol (yellow) 612A
+
-
HSO,'-
ANALYTICAL CHEMISTRY, V O L
The presence of an added inorganic anion causes a shift in the equilibrium between substrate and product. Moreover, the anion in both cases above is believed to act as a competitive inhibitor. A schematic representation of the anion-monitoring system is shown in Figure 12. Because of the mode of operation, the system is relatively unaffected by column flow rate, temperature, or even a relatively wide range of molar concentrations of the substrate. Operating the system with a relatively high salt concentration eliminates the nonspecific anionic ef46, N O . 7, J U N E 1 9 7 4
fects, and the system responds only to the anion of interest. Immobilized enzymes in many different forms and systems can be used for a broad range of analytical purposes. For example, this limited report has covered: enzymes immobilized in membranes closely associated with ion-specific electrodes; organic ion-specific electrodes that monitor organic species, i.e., choline used with soluble and insoluble enzymes; immobilized enzymes in plug flow or tubular reactors interfaced with readout systems. In addition, there are many other types of immobilized enzyme analytical systems. For instance, Messing ( 3 1 )reported an assay of glucose by using glucose oxidase and differential conductivity. Messing's method is based on the conversion of a relatively nonconducting species of glucose into a more conductive species-gluconic acid. Similarly, urease and differential conductivity could be used to measure the conversion of urea to ammonia and Con. It is not within the scope of this review to discuss the range of industrial and therapeutic applications for immobilized enzymes. These ureas have been covered in depth elsewhere in the literature ( 4 , 17, 20, 3 2 ) . Summary Despite the rather extensive research effort being spent in enzyme technology, it is still regarded by many as "a solution in search of a problem." It is probably true that the real opportunities for immobilized enzyme technology lie 5 or 10 years ahead. Certainly, the emphasis in this field today-both in governmentfunded research and in industry-is on its use in large-scale systems for the food, beverage, and pharmaceutical fields. With respect to analytical applications, several companiesnotably, Corning Glass Works. Leeds, and Northrup, and Technicon-are developing instruments that utilize immobilized enzymes. Indeed, one, Yellowsprings Instrument Co., is already marketing a glucose analyzer which uses glucose oxidase trapped in a membrane on a platinum electrode-a system based on the approach recently described by Clark (24). Owing principally to their high degree of reaction selectivity and their speed, enzymes, particularly immobilized enzymes, hold considerable promise in a range of process-related fields, not the least of which is analytical instrumentation. References (1) J. M . Nelson and E. G. Griffin, J.
Amer. Chem. Soc.. 38, 1109 (1916).
EM Reagents@
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E. MERCK, DARMSTADT, GERMANY
r-
The right way to optimal separations
Silica gels 40, 60, 100
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diameter)
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Aluminum Oxide Type E a n d T Kieselguhr Merckosorbc
Cellulose ' Cellulose ion exchangers
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EM Laboratories, Inc., associate of E. Merck, Darmstadt, G e r m a n y 500 Executive Boulevard, Elmsford, New York 10523 Tel: 914/592-4660 CIRCLE 6 4 ON READER SERVICE CARD A N A L Y T I C A L CHEMISTRY, V O L . 46, N O . 7, J U N E 1974
613A
The IR:
Hou)ard H . W e e t a l l ( r i g h t ) and L . S . Hersh are s h o u n i n t h e process of bonding heparin t o polyester t u b i n g a t Corning's research laboraton ( 2 ) 1. Grubhofer and L. Schlecth, Hoppe Seyler's 2. Physiol. ('hem.. 297, 108
Basic Specifications Wavenumber
Range 400-4000cm" (25-2.5rm) Expansion factor lX, 5X Scan times 3,6, 25, 55 minutes
Transmission
Scale Expansion Presentation
0-1 00%
0.8-10X, continuously variable Integral strip chart recorder with 50 charts, ruled with a 15X52cm grid or continuous 15cm wide grid.
Performance
Resolution Normal slit Droaram Narrow slit program Photometric accuracy Photometric reDeatabilitv Beam balance
4cm" at 3000cm" 2.5cm.l at 1000cm2.5crn.l at 3000cm-' 1 5cm" at 1000cm'I
(1954).
F.
(3) Grubhofer and L. Schlecth. iVnturu,zssenschajten, 40, 508 (1953). (4) 0. R.,Zahorsky. "Immobilized E n zymes, CRC Press, Cleveland. Ohio, 1973. (5) A. D. McLaren and L. Packer. Adt,an. Enz\.mo/. Reiat. A r e a s Moi. Bioi.. 33,
245 (1970).
(61 C. A. Zittle, ibid.,14, 319 (1953). ( 7 ) R. A. Messing. J , Amer. Chem. SOC..
91,2370 (1970). (8)R. A. Messing, Enz? moiogia. 38,370 (1970). (9) R. Haynes and K . A. Walsh. Biochem Biophys. Res. Commun., 36, 235 (1969). (10) W.Gaffield. Y.Tomimatsu. A. C. Olson. and E. F . Jansen. Arch. Biochem. Bioph>,.s.. 157,405 (1973). (11) F%-.L.Stanleyand A. C. Olson. U.S. Patent 3.736,231 (1973). (12) P. Bernfeld and J. Wan. Science. 1.12, 678 (1963). (13) T. M . S.Chang. i b i d . . 146,524 il9tXi . / _ _ _ .
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Solid state pneumatictype with KRS-5 window.
What you get it for: $7995
A North American Philips Company 750 South Fulton Avenue Mt. Vernon, NY 10550
(14) A. Bar-Eli a n d E. Katchalski. 'Vaturc. 188,856 (1960). (15) H. H . b'eetall, Science. 166.615 (1969). (16) G. R. Stark. "Biochemical Aspects of
(23) G. Guilbault. in "Enzyme Engineering." L. B. Wingard, J r . . Ed.. p p 361-76. Wiley. New York. N.Y., 1972. (24) L. C. Clark, J r . . Ed., i b i d . , pp377-
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(25) G. Baum and F. Ward. A n n / . Biochem., 42,487 (1971). (26) G. Bau,m. F. LVard. and S. Yaverbaum. Clin. Chem. Acta. 36,406 (1972). ( 2 7 ) M . K. LV. Weihel. LV. Dritschilo. H . J. Bright. a n d A. E . Humphrey. Anai. Biochem., 52,402 (197.3). (28) D. J . Inman a n d W. E . Hornby, H i o chem. J.. 129,255 (1972). (29) A. L. Chaney and E. P . Marhach. Clin. Chem.. 8, 130 (1962). (30) H . H. LVeetall and M . A. .Jacobson. Proc. IV IFS Ferment. Tech. Today.
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mess ( 3 i ) H. H. LVeetall. Food Prod. Develop.. Part I. April 1973: Part 11, M a ) 1973 (33) H . H. A'eetall and N.B. Havei\ala. "Enzyme Engineering." Wiley. New 1972. York. N.Y.. '34) H . H . Weetall. N.B. Havewala. 1%'. Pitcher. Jr.. C. C. Detar. W . P. Van. and S. Yaverbaum. Biotr~ch.Hiocne.. 16,
Reactions on Solid Supports." Academic Press. S e w York. N.Y., 1971. (17) H . H. Weetall and R . A. Messing. in "The Chemistry of Biosurfaces." M. L. Hair, Ed.. Yo1 11, Marcel-Dekker, New York, N.Y.. 1972. (181 H . H . CVeetall. Separ Purification Methods, 2, 199 (1973). (19) L. Goldstein. Y. Levin, a n d E. K a t chalski. Biochemistr). 3, 1913 (19641. (20) L. B. Wingard. Jr.. E d . , "Enzyme Engineering S y m . 3," LViley. New York. N.Y.. 1972. (21) I. H . Silman and E. Katchalski. A n n . R e v . Hiochcm.. 35,873 (1966). ( 2 2 ) S..J. Updike a n d G . P. Hicks. ture. 214,986 (1967).
295 (1974).
Howard H. Weetall is a senior research associate in biochemistry in Corning's Research and Development Division. H e joined the company in 1967 a s a research biochemist, was appointed a research associate in 1971, and was given his present position in 1973. Author of many technical articles, Weetall holds BA and MA degrees from the University of California a t Los Angeles.
CIRCLE 191 ON READER SERVICE CARD
A N A L Y T I C A L CHEMISTRY
VOL
46,
NO 7 JUNE 1974
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615A
