Appendix - Springer978-1-60327-407-4/1.pdf · is usuall y any need, especiall y if you memoriz e the log of 2 and 1.25 (0.3 and0.1).(SeeAHomemade Lo g Tabl e fefow>.)Youcanevenforgetwhether

Appendix Acid-Base Made Easy

Some Definitions

LOGARITHM (TO THE BASE 10)

A logarithm is an exponent. It is a number that, if used as an exponent to 10, will give the number whose log it is.

EXAMPLES

103=1000 log 1000 = 3 10"3 = 0.001 log 0.001 = - 3

1003 = 2 log 2 = 0.3 10-o.3 = i/2 = o.5 log 0.5 = -0.3

Remember, log N + log M = log N x M, log N - log M = log N/M, log 10"N = log 1/10N, log VN = 1/2 log N. (See also A Homemade Log Table at the end of this section.)

P H

pH is the negative logarithm of the hydrogen ion concentration (i.e., pH = -log [H+] or 10"pH [H+]). (Note concentration is always molar.)

EXAMPLES

[H+] = 0.1M 0.1 = 10^ pH=l [H+] = 0.0001M 0.0001 = 10 "4 pH = 4 [[H+] = 0.5Af 0.5 = 5xlO"1 pH = -(0.7-1) = 0.3 [[H+] = 0.0002M 0.0002 = 2 x 10 ~ 4 pH = - (0.3 - 4) = 3.7 [H+] = 5 x 10"9M pH = - (0.7 - 9) = 8.3

ACID

Acid is any compound that can lose a hydrogen ion.

EXAMPLES

CH3COOH, CH3CH2OH, CH3NH3+, NH4

+

Note "strong acids," such as HCl, really do not fit this definition, see under Conjugate Base.

BASE

Base is any compound than can gain a hydrogen ion.

365

366 Passonneau and Lowry

EXAMPLES

CH3COO-, CH3CH2O-, CH3NH2, NH3

CONJUGATE BASE (OR ACID)

This is the base (or acid) in the following general equation:

acidM>ase + H+ [Eq.A-l]

i.e., every acid has its conjugate base (and every base has its conjugate acid). Strong acids and bases, such as HCl and NaOH, really do not fit this formulation. HCl in solution has already lost its H+ (i.e., it is completely dissociated and can be written H+C1"). NaOH is really Na+OH~ and the real base is OH", which qualifies because it can gain a H+:

OH + H * ^ H 2 0 [Eq.A-2]

BUFFER

Buffer is a mixture of a conjugate acid and base. It is a buffer (i.e., a pH buffer), because if H+ is added most of H+ will combine with the base. Eq. (A-l)], lessening the increase in H+ concentration that would otherwise result. Similarly, if H+ is removed from such a mixture, more will be formed from the acid [Eq. (A-1)], lessening the decrease in H+ concentration that would otherwise result. P.Ks AND THE HENDERSON-HASSELBALCH EQUATION

The more readily an acid loses H+, the stronger an acid it is. This strength is measured by its dissociation constant

[base][H+] [B][H+] K= —7—rrr— = rA1 [Eq. A-3]

[acid] [A] L M J When the acid is 50% dissociated, base and acid are equal and K = [H+]. For example, the dissociation constant of acetic acid is 2.5 x 10~5M. Therefore, in a mixture of equal molar concentrations of acetic acid and sodium acetate [H+] = 2.5 x 10"5M or pH = - (0.4 - 5) = 4.6.

Similarly, the dissociation constant of NH4+ is 5 x 10"10. Therefore, in

a mixture of equal molar concentrations of ammonium hydroxide (NH3 • H20) and NH/Cl", [H+] = 5 x 10"10 or pH = - (0.7-10) = 9.3.

Because of this relationship between AT and [H+], and by analogy with pH, the negative log o/K is calledpK. It is convenient to replace K and [H+] in Eq. [A-3] with pH and pK by taking the log of each side.

Appendix 367

logK = log 1 1 1 0 0 = log 111+ log [H+] r c

[A] [A] ^ - A"41

rearranging:

-log[H1 = -logK + logMorpH = pK + log[Bl r F A _ [A] [A] m' A ° J

This is the famous Henderson-Hasselbalch equation. This equation was originally written:

pH = pK + log [SaUl [ E q A-6] [Acid]

This was before Bronsted proposed the definition of acids and bases given above. Equation [A-6] is harder to use than Eq. [A-5], and is not quite exact.

From the pK of an acid and the Henderson-Hasselbalch equation, it is easy to calculate the pH for any ratio of acid to base.

Base/acid 100 1/100 20 1/20 10 1/10

PH pK + 2 pK-2 pK+ 1.3 ptf-1.3 pK+l pK-l

Base/acid 2 1/2 1 200 5000 10,000

PH pK + 0.3 pK-03 pK pK+23 ptf+3.7 pK+4

It is easy to make this calculation in your head, as accurately as there is usually any need, especially if you memorize the log of 2 and 1.25 (0.3 and0.1).(SeeAHomemade Log Table fefow>.)Youcanevenforgetwhether it is base/acid or acid/base in the H-H equation if you simply remember that, if base predominates, the pH is on the basic side of the pK, and if acid predominates, the pH is on the acid side of the pK.

However, if you prefer, Table 1 makes the calculation for you and gives the percentage of the buffer that is in the basic form. Notice it takes three times more acid or base to make a pH change of 0.1 unit near the pK than it does 1 pH unit away. At 1.3 units from pK, a 1% shift from acid to base makes a 0.1 unit pH change. Thus, the useful pH range is limited to about 1 pH unit on either side of the pK.


Table 1 Base:Acid Ratios to Give Desired pH

pH pK+ 1.0 pK+0.9 pK+O.S pK + 0.1 pK+0.6 pK+0.5 p£+0.4 pK+03 pAT+0.2 pK+O.l p£+0.0

MAKING

Base acid 10 8 6.3 5 4 3.2 2.5 2 1.6 1.25 1.0

A BUFFER

Base as % ofA + B

91 89 86 83 80 76 71 67 61 56 50

pH p*-1.0 pK-0.9 ptf-0.8 ptf-0.7 pK-0.6 pK-0.5 pK-OA pAT-0.3 pK-0.2 pAT-0.1 pAT-0.0

Base acid

~~10 8 6.3 5 4 3.2 2.5 2 1.6 1.25 1.0

Base as % ofA + B

9 11 14 17 20 24 29 33 39 44 50

A buffer mixture can be made by either (a) mixing the free acid or base with its salt, or (b) adding a calculated amount of strong acid or strong base to the free base or free acid. For example, a pH 4.6 buffer can be made by making the solution 50 mM in acetic acid and 50 mM in sodium acetate, or it can be made by making the solution 100 mMin acetic acid and 50 mM in NaOH. This ignores the fact that some H+ had to be produced (10"4 6M) from the acetic acid. Therefore, the base (acetate) is in fact slightly greater than the added sodium or NaOH. In this case, the difference is only 1 part in 2000. Only below pH 3 and above pH 11, and with low buffer concentrations, does this ever become significant.

MIXTURE OF Two OR MORE ACID-BASE PAIRS

The H-H equation must apply simultaneously to all acid-base pairs in a given solution, i.e.:

[Bi] [B2] pH = pK! + log ^ = pK2 + log p ^ etc. [Eq. A-7]

Take as an example a mixture of acetic acid, acetate (pAT=4.6), Tris+, and Tris base (pK = 8.1). At pH 7:

log [BJ/tAJ = log [Ac]/[HAc] = 2.4 = log 500/1 and A g ]

log [BJ/[AJ = log [Tris]/[Tris*] = -1.1 = log 1/12.5

Appendix 369

DISSOCIATION CONSTANT OF H20

There is one acid that must not be forgotten; this is H20. It is a very weak acid; its pK is 15.7. That means that:

rtrifOHl = 10157 M and pH = 15.7 + log FOH1 [Eq. A-9] [H20] [H201

Because the concentration of H20 is practically constant in most aqueous solutions (55.5M), it has become customary to merge its concentration with the dissociation constant:

[H1[OH] = 55.5 x 10157 = 10"14 [Eq. A-10]

In the logarithmic form, this becomes:

pH + pOH = 14 or pOH = 14 - pH ^ A*nJ

where pOH is the negative log of [OH-]. PH OF FREE ACIDS AND BASES

The pH of a weak acid (or base) in the absence of added conjugate base (or acid) is easily calculated in all practical situations. When an acid is dissolved in H20, A<-» B + H+ (i.e., B = H+). Equation [A-3] rearranged becomes [B][H+] =£[A], or in this case [H+]2 = K(A)9 and [H+] = V#[A], or:

pH = l/2(pK-log[A]) [Eq.A-12]

For example, O.IM acetic acid has pH = 1/2(4.6 + 1) = 2.8. A 0.01M solution (-log [A] = 2) would have a pH of 3.3. The pK of NH4

+ is 9.3. A 50 mM solution of NH4C1 has pH = 1/2(9.3 + 1.3) = 5.3. The pH of a IM solution would be 4.65.

When a free base is dissolved in H20, it removes H+, forming hydroxyl ion: B + H 2 O H A + OH". Here [A] = [OH"] = 10"14/[H+] (see above). Equation [A-3] rearranged becomes [H+]/[A] = K/[B], Substituting for [A]:

[H*]2/10-14 =K/[B], [H*]2 = 1014 K/[B], and [H+] = Vl014K/[B], or

pH= 1/2(14 + pK + log [B]) ^ A"13l

For example, 0.2M Tris base has pH = l/2( 14 + 8.1 - 0.7) = 10.7. A 0. IM sodium acetate solution has pH = 1/2(14 + 4.6 - 1) = 8.8. (An easy way to remember Eqs. [A-12] and [A-13] is that an acid has a pH halfway


between its ptfand the pH it would have if it were a strong acid, and a base has a pH halfway between its pK and the pH it would have if it were a strong base.)

Equations [A-12] and [A-13] are not strictly true, because they ignore the decrease in A or in B required to form H+ in one case and OH" in the other. The difference is exceedingly small, except with low concentrations of acids having pKs below 3 or bases with pKs over 11.

SOME COMPLICATIONS WITH RKS

We have been talking about pKs and dissociation constants as though they were invariant. Actually, they are affected by temperature and salt concentration (ionic strength). Increasing temperature and increasing ionic strength increase the dissociation constants (decrease the pKs). Tables of pKs are often calculated for 25° for "infinite dilution" (zero ionic strength). An observed pK is therefore, always lower than these "true" pKs, and should properly be called an apparent pK and written pK.

Ionic strength has a much greater effect if the dissociation involves a separation of charges than if it does not. Consider the three dissociations:

RNH3+^RNH2+H+

[Re. A-l] RCOOH % RCOO + H*

RP02H % RP022 + H+

Ionic strength will have little effect in the first case, a moderate effect in the second, and a large effect in the third. (Increasing ionic strength by increasing the dielectric constant of the solution, shields the charges from each other, making it easier for them to come apart.)

As examples, the true pK of acetic acid is given as 4.76, but a 0.1M acetate buffer has a plC near 4.6. The pK2 of a phosphate buffer is 7.2, a 0. IM phosphate buffer has a pKj of 6.8, and in a IM phosphate buffer the pK2' is around 6. (The pK3 is even more sensitive to ionic strength.) True pKs are given in Table 2, and in some cases, ionic strength effects may need to be taken into account.

There is one final complication in regard to pKs. We have been considering NH4

+, for example, as an acid with pK = 9.3 (i.e., a dissociation constant of 5 x 10~10. Unfortunately, in preBronsted days, NH3 was regarded in solution as NH4OH, which dissociated as NH4OH <-> NH4

+ +

Appendix 371

OH". Measurements of OH" concentration indicated a dissociation constant of 2 x 10"5; or pK=4.7. To distinguish these, one is written pKa (for the acid dissociation constant); the other is written pKb (for the "base dissociation constant"). It is clear that, since pOH + pH = 14, pKa + pKb

= 14. pKbs are still to be found in handbooks, often written simply as pK, and it is not always clear whether p£a or pKb is meant.

A HOMEMADE LOG TABLE

All the logarithms needed for practical acid-base aspects of most analytical work can be carried in your head, or figured out quicker than you can look up a log table. All you need to remember is that the log of 2=0.3 (actually 0.30103) and the log of 1.25 = 0.1 (actually 0.0969). Knowing the log of 2, it is clear that log 4 = 0.6 and log 8 = 0.9. Similarly, log 5 = 0.7 (i.e., log 10 - log 2), and log 2.5 = 0.4 (i.e., log 5 - log 2). We are only missing 0.2,0.5, and 0.8, which we obtain as the following tabulation:

log 1 = 0.0 log 1.25 = 0.1 (memory) log 1.6 = 0.2 (log 2-log 1.25)

log 2 = 0.3 (memory) log 2.5 = 0.4 (log 10-log 4) log 3.2 = 0.5 (log4-log 1.25)

log 4 = 0.6 (log 2 +log 2) log 5 = 0.7 (log 10-log 2)

log 6.3 = 0.8 Gog 5 +log 1.25) log 8 = 0.9 (log 4 +log 2)

log 10 =1.0

If you need to be closer than 0.1 pH unit, you can interpolate from this, e.g., log 9 is close to 0.95 (0.954). This list and the Henderson-Hasselbalch equation, both in your head, will save time and, more importantly, avoid blunders. STATISTICAL SHORTCUTS

This section is aimed at the "casual statistician" (Snedecor). Rarely, for his purposes, does the situation require or justify elaborate statistical treatment.

We are concerned here only with means, standard deviations, standard errors (of the mean), and standard errors of differences.

First, the orthodox calculations: Standard deviation (St. dev.) = Vsum of dev2 ^Eq A . 1 4 j

n - 1


where dev is the individual deviation from the means and n is the number of samples.

[Eq. A-15]

Standard error (of the mean) (SE or SEM) = Vsum of dcvT=st dev n(n - 1) Vn

i - T [Eq.A-16] Standard error of the difference (SED) = vSEj2 + SE/

There is a longer way of calculating SED, but it has no clear advantage. Now the shortcuts.

1. For most purposes, calculation of SE to within 10% is all that is worthwhile. For example: 110 ± 10 (SE) says that there is a 1 out of 3 chance that the true mean lies outside of the range 90-110. What virtue could there possibly be in calculating that the SE is actually 10.3?

2. Conversely, calculation of the mean to closer than 10% of the SE is seldom worthwhile. In the previous example, what possible use would there be in calculating that the average is actually 110.4? If, however, the standard error was 1 instead of 10, it might be useful to calculate the mean more closely, say ± 0.1.

3. An approximate value for the SE is: SE = range of values ^ A"17]

n For example, if there are 10 values ranging from 80 to 120, SE = 40/10 = 4. This gives a surprisingly close estimate of the SE (usually within 15%). It is a valuable check for errors in more exact calculations. (Note, however, that with n > 12 or 13, this gives erroneous small estimates of SE).

4. Similarly, because SE = SDNn:

st. dev. = SEVn = range [Eq. A-18] Vn

5. For more orthodox calculations of means, SEs, and SDs, individual values and individual deviations from the mean can usually be rounded off with great saving of time. What guidelines are there to safe rounding off?

It is usually completely safe to round off individual values to within 10% of the range. For example, there are 10 numbers ranging from 32.2 to 43.6. The range is 11.4. Therefore, the numbers can be rounded off to the nearest whole number. No number will be changed by more than 0.5. This cannot increase the range by more than twice 0.5 or 1 (i.e., 9%).

Appendix 373

Therefore, this will not increase the SE or the SD by more than 9%, as the above shortcut method for calculating these statistics indicates.

Rounding off to within 10% of the range will have a completely negligible effect on the mean. Thus, in the above case, the average number is changed by only 0.25, and changes are randomized plus and minus.

Table 2 Dissociation Constants for Acids and Bases Used in Making Buffers

Acid or base

Oxalic acid, Kj P-glycerophosphoric, Kt

Histidine, K, Maleic, Kt

Phosphoric, Kt

Aspartic, Kj Glutamic, K, Arsenic, K, Lysine, Kx

Glycine, Kt

Phthalic, Kt

Tartaric, IQ Citric, Kj Fumaric, Kt

Glycylglycine, Kj Malic, Kj Formic Aspartic, K2

Lactic Barbituric Glutamic, K2

Tartaric, K2

Succinic, Kt

Oxalic, K2

Fumaric, K2

Citric, K2

Acetic Malic, K2

Benzoic Phthalic, K2

Succinic, K2

Maleic, K2

Pyrophosphoric, K3

Uric Citric, K3

Histidine, K2

pK> 1.21 1.47 1.73 1.83 1.96 2.14 2.14 2.25 2.25 2.35 2.92 3.00 3.05 3.10 3.10 3.40 3.76 3.87 3.87 4.00 4.09 4.13 4.13 4.22 4.53

4.75 4.76 5.00 5.20 5.57 5.57 5.75

5.77 5.83 6.00 6.10

Acid or base

4-Phenylimidazole MES** P-glycerophosphoric, K2

Cacodylic

2-Phenylimidazole 4-Hydroxyimidazole Arsenic, K2

Imidazole Phosphoric, K2

BESW

MOPSc

2,4,6-Collidine TES** HEPESeA

4-Methyimidazole Triethanolamine 2-Methyimidazole Glycylglycine, K2

Tri/ Tricine*' Pyrophosphoric, K2

Hydrazine, K2

2,4-Dimethylimidazole 2-Amino-2-methyl-1.3

propanediol Lysine Histidine, K3

Ammonium hydroxide Glutamic acid, K3

Glycine, K2

Aspartic acid, K3

2-Amino-2-methyl-l -propanol

Carbonic acid, K2

Lysine, K3

Arsenic, K3

Phosphoric, K3

PKa 6.13 6.15 6.19 6.19

6.53 6.53 6.77 7.07 7.12 7.15 7.2 7.40 7.5 7.55 7.65 7.9 8.00 8.07 8.1 8.15 8.22 8.23 8.48

8.78 8.92 9.14 9.23 9.43 9.75 9.75

9.90 10.36 10.54 11.6 12.24

aMES, 2-(N-morpholino)ethanesulfonic acid. *BES, tyN-fcw(2-hydroxyethyl)-2-aminoethanesulfonic acid. cMOPS, 3-N-(Morpholino)propanesulfonic acid. *TES, N-fris(hydroxymethyl)methyl-2-aminosulfonic acid. 'HEPES, N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid. fTris, rrw(hydroxymethyl)aminoethane. *Tricine, AT-rrw(hydroxymethyl)methylglycine. *At 20°, Good et al., 1966. 'Good, 1962. 3 4

Appendix 375

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TableS An Abbreviated List of Atomic Weights

Aluminum Antimony Arsenic Barium Bismuth Boron Bromine Cadmium Calcium Carbon Cesium Chlorine Chromium Cobalt Copper Fluorine Gold Hydrogen Iodine Iron Lanthanum Lead Lithium Magnesium Manganese Mercury Molybdenum Nickel Nitrogen Oxygen Phosphorus Platinum Potassium Selenium Silicon Silver Sodium Strontium Sulfur Tellurium Tin Tungsten Uranium Zinc

Al Sb As Ba Bi B Br Cd Ca C Cs CI Cr Co Cu F Au H I Fe La Pb Li Mg Mn Hg Mo Ni N 0 P Pt K Se Si Ag Na Sr S Te Sn W U Zn

27.0 121.8 74.9

137.4 209.0

10.8 79.9

112.4 40.1 12.0

132.9 35.5 52.0 58.9 63.5 19.0

197.0 l.(X

126.9 55.8

138.9 207.2

6.9 24.3 54.9

200.6 96.0 58.7 14.0 16.0 31.0

195.1 39.1 79.0 28.1

107.9 23.0 87.6 32.1

127.6 118.7 183.9 238.1

65.4

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3. Kerr, S. E. and Ghantus, M. (1937) The carbohydrate metabolism of the brain. III. On the origin of lactic acid. J. Biol. Chem. 117,217-225.

4. Ferrendelli, J. A., Gay, M. H., Sedgwick, W. G., and Chang, M. M. (1972) Quick-freezing of the murine CNS: Comparison of regional cooling rates and metabolite levels when using liquid nitrogen or Freon-12. J. Neurochem. 19, 979-987.

5. Nelson, S. R., Lowry, O. H., and Passonneau, J. V. (1966) in Head Injury Conference Proceedings (Caveness, W. F. and Walker, A. E., eds.), Lippincott, Philadelphia, PA, p. 444.

6. Bessey, O. H., Lowry, O. H., and Love, R. H. (1949) The fluorometric measurement of the nucleotides of riboflavin and their concentration in tissues. J. Biol. Chem. 180,755-769.

381


7. Burch, H. B., Bradley, M. E., and Lowry, O. H. (1967) The measurement of triphosphopyridine nucleotide and the role of hemoglobin in producing erroneous triphosphopyridine nucleotide values. J. Biol Chem. 242,4546-4554.

Chapter 5 1. Cha, S. and Cha, C.-J. M. (1965) Kinetics of cyclic enzyme systems. Mol

Pharmacol 1,178,179. 2. Hintz, C. S., Chi, M. M.-Y., and Lowry, O.H. (1980) Correcting potential de

fect in an enymatic cycle for NADP. Anal Biochem. 128,186-190. 3. Lowry, O. H„ Passonneau, J. V., Schulz, D. W., and Rock, M. K. (1961) The

measurement of pyridine nucleotides by enzymatic cycling. J. Biol Chem. 236,2659-2756.

4. Chi, M. M.-Y., Lowry, C. V., and Lowry, O. H. (1978) An improved enzymatic cycle for nicotinamide adenine dinucleotide phosphate. Anal Biochem. 89,119-129.

5. Kato, T., Berger, S. J., Carter, J. A., and Lowry, 0. H. (1973) An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. Anal Biochem. 53,86-97

6. Breckenridge, B. McL. (1964) The measurement of cyclic adenlyate in tissues. ProcNatl Acad. Sci. USA 52,1580-1586..

7. Goldberg, N. D., Dietz, S. B., and O'Toole, A. G. (1969) Cyclic guanosine 3\5' monophosphate in mammalian tissues and urine. J. Biol. Chem. 224,4458-4466.

Chapter 6 1. Kornberg, A. and Pricer, W. E., Jr. (1951b) Enzymatic phosphorylation of

adenosine and 2,6-diaminopurine riboside. J. Biol Chem. 193,481-495. 2. Pfleiderer, G., Grein, L., and Wieland, T. (1955a) Specific determination of L-

alanine and L-glutamine by means of glutamic-pyruvic transaminase. Ann. Acad. Sci. Fenn. Ser. A2, 60, 381-388.

3. Pfleiderer, G., Gruber, W., and Wieland, T. (1955b) Eine enzymatische bestimmung der L-asparaginsaure. Biochem. Z. 326,446-450.

4. Kornberg, A. (1950) Reversible enzymatic synthesis of diphosphopyndine nucleotide and inorganic pyrophosphate. /. Biol. Chem. 182,779-793.

5. Moellering, H. and Gruber, G. (1966) Determination of citrate with citrate lyase. Anal. Biochem. 17, 369-376.

6. Thorn, W., Pfleiderer, G., Frowein, R. A., and Ross, I. (1955) Stoffwechselvor-gange im Gehirn bei akuter Anoxie, akuter Ischamie und in der Erholung. Pflueger'sArch. Gesamte Physiol. Menschen Tiere 261,334-360.

7. Slein, M. W. (1950) Phosphomannose isomerase. J. Biol. Chem. 186,753-761. 8. Slater, E. C. (1953) Spectrophotometric determination of fructose-1 ̂ -diphos

phate, hexose monophosphates, adenosinetriphosphate and adenosine-diphosphate. Biochem. J. 53,157-167.

9. Vishniac, W. and Ochoa, S. (1952) Fixation of carbon dioxide coupled to photochemical reduction of pyridine nucleotides by chloroplast preparations. J. Biol. Chem. 195, 75-96.

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10. Slein, M. W., Cori, G. T., and Cori, C. F. (1950) A comparative study ofhexo-kinase from yeast and animal tissues. /. Biol. Chem. 186,763-780.

11. Paladini, A. C, Caputto, R., Leloir, L. F., Trucco, R. E., and Cardini, C. E. (1949) The enzymatic synthesis of glucose- 1,6-diphosphate. Arch. Biochem. 23,55-66.

12. Albers, R. W., Koval, G. McKahnn, G„ and Ricks, D. (1961) in Regional Neu-rochemistry (Kety, S. S. and Elkes, J., eds.), Pergamon, Oxford, p. 340.

13. Bublitz, C. and Kennedy, C. P. (1954) Synthesis of phosphatides in isolated mitochondria III. The enzymatic phosphorylation of glycerol. /. Biol Chem. 211,951-961.

14. Ochoa, S. (1948) Biosynthesis of tricarboxylic acids by carbon dioxide fixation. III. Enzymatic mechanisms. /. Biol. Chem. 174,133-157.

15. Noll, F. (1966) Methode zur Quantitativen Bestimmung von L(+)-Lactatmittels Lactat-Dehydrogenase und Glutamat-Pyruvate Transaminase. Biochem. Z. 346, 41-49.

16. Kubowitz, F. and Ott, P. (1943) Isoliering und Kristallisation eines Garungs-fermente aus Tumoren. Biochem. Z. 314,94-117.

17. Strominger, J. L., Maxwell, E. S., and Kalckar, H. M. (1957) Meth. Enzymol. 3,974.

18. Strominger, J. L. (1955) Enzymic synthesis of guanosine and cytidine triphosphates: A note of the nucleotide specificity of the pyruvate phosphokinase reaction. Biochim. Biophys. Acta 16,616,617.

19. Noda, L. and Kuby, S. A. (1963) Meth. Enzymol. 6,223. 20. Lowry, O. H., Schulz, D. W., and Passonneau, J. V. (1964) Effects of

adenylic acid on the kinetics of muscle phosphorylase a. J. Biol. Chem. 239, 1947-1953.

21. Barbehenn, E. K., Law, M. M-Y., Brown, J. G., and Lowry, O. H. (1976) Measurement of 5'adenylic acid by stimulation of phosphorylase a. Anal. Biochem. 70, 554-562.

22. Lienhard, G. E. and Secemski, 1.1. (1973) P1,P5-Di(adenosine-5')penta-phos-phate, a potent inhibitor of adenylate kinase. /. Biol. Chem. 248,1121-1123.

23. Lust, W. D„ Feussner, G. K., Barbehenn, E. K., and Passonneau, J. V. (1981) The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts. Anal. Biochem. 110,258-266.

24. Manchester, J. K., Chi,M. M.-Y., Carter, J., Pusateri, M.E.,McDougal,D. B., and Lowry, O. H. (1990) Measurement of 2-deoxyglucose and 2-deoxyglucose-6 phosphate in tissues. Anal. Biochem. 185,118-124.

25. Baranowski, T. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), rev. ed., vol. 7, Academic, New York, p. 63.

26. Velick, S. F. and Furfine, C. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), vol. 7, Academic, New York, p. 243.

27. Massey, V. and Alberty, R. A. (1964) Ionisation constants of fumarase. Biochim. Biophys. Acta 13,354-359.

28. Ray, W. J. Jr. and Roscelli, G. A. (1964) A kinetic study of the phosphogluco-mutase pathway. J. Biol. Chem. 239,1228-1236.


29. Frieden, C (1959a) Glutamic dehydrogenase II. The effect of various nucleotides on the association-dissociation and kinetic properties. 7. Biol. Chem. 234, 815-820.

30. Frieden, C. (1959b) Glutamic dehydrogenase III. The order of substrate addition in the enzymatic reaction. J. Biol Chem. 234,2891-2896.

31. Matschinsky, F. M. (1964) Personal communication. 32. Passonneau, J. V. and Lauderdale, V. R. (1974) A comparison of three meth

ods of glycogen measurement in tissues. Anal Biochem. 60,405-412. 33. Lust, W. D., Passonneau, J. V., and Crites, S. K. (1975) The measurement of

glycogen in tissues by amylo-a-l,4-a-l,6-glucosidase after the destruction of preexisting glucose. Anal Biochem. 68,128-131.

34. Passonneau, J. V. and Rottenberg, D. A. (1973) An assessment of methods for measurement of glycogen synthetase activity including a new direct one-step assay. Anal. Biochem. 51,405-412.

35. de Azeredo, F. A. M., Feussner, G. K., Lust, W. D., and Passonneau, J. V. (1979) An enzymatic method for the measurement of GTP and GDP in biological samples. Anal Biochem. 95,512-519.

36. Kaufman, F. and Alivisatos, S. G. A. (1955) Purification and properties of the phosphorylating enzyme from spinach. /. Biol Chem. 216,141-152.

37. Cha, S. and Parks, J. E., Jr. (1964) Succinic thiokinase. II Kinetic studies; initial velocity, product inhibition, and effect of arsenate. J. Biol. Chem. 239, 1968-1977.

38. Olson, J. A. and Anfinsen, C. B. (1953) Kinetic and equilibrium studies on crystalline L-glutamic acid dehydrogenase. /. Biol. Chem. 202, 841-856.

39. Hintz, C. S., Chi, M. M.-Y., Fell, R. D., Ivy, J. L., Kaiser, K. K., Lowry, C. V., and Lowry, O. H. (1982) Metabolite changes in individual rat muscle fibers during stimulation. Am. J. Physiol. 242, C218-C228.

40. Krebs, H. A. (1953) Equilibria in transamination systems. Biochem. J. 54,82-86. 41. Fawaz, E. N., Roth, L., and Fawaz, G. (1966) The enzymatic estimation of

inorganic phosphate. Biochem. Z 344,212-214. 42. Pontremoli, S., de Flora, A., Grazi, E., Mangiarotta, G., Bonsignore, A., and

Horecker, B. L. (1961) Crystalline D-gluconate-6-phosphate dehydrogenase. J. Biol. Chem. 236,2975-2980.

43. Outlaw, W. H., Jr. and Lowry, O. H. (1979) Measurement of 10~7 to 10"12 mol of potassium by stimulation of pyruvate kinase. Anal Biochem. 92,370-374.

44. Cook, G. A., O'Brien, W. E., Wood, H. G., King, M. T., and Veech, R. L. (1978) A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues. Anal. Biochem. 91,557-565.

45. Jones, M. G. K., Outlaw, W. H., Jr., and Lowry, O. H. (1977) Enzymic assay of 10"7 to 10~14 moles of sucrose in plant tissues. Plant Physiol. 60, 379-383.

Chapter 7 1. Henriksson, J., Chi, M. M.-Y., Hintz, C S., Young, D. A., Kaiser, K. K, Salmons,

S., and Lowry, O. H. (1986) Chronic stimulation of mammalian muscle: Changes in enzymes of six metabolic pathways. Am. J. Physiol. 251, C614-C632.

References 385

2. Cole, B. R, Hays, A. E., Boylan, J. B., Burch, H. B., and Lowry, O. H. (1982) Distribution of enzymes of adenylate and guanylate metabolism in rat nephron. Am. 7. Physiol 243,F349-F355.

3. Chi, M. M.-Y., Hintz, C. S., Coyle, E. F., Martin, W. H. Ill, Ivy, J. L, Nemeth, P. M., Holloszy, J. O., and Lowry, O. H. (1983) Effects of detraining on enzmes of energy metabolism in individual human muscle fibers. Am. J. Physiol 244, C276-C287.

4. Chan, A. W. K., Perry, S. G., Burch, H. B., Fagioli, S., Alvey, T. R., and Lowry, O. H. (1979) Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats. 7. Histochem. Cytochem. 27,751-755.

5. Burch, H. B., Lowry,O. H., Kuhlman, A. M., Skerjance, J.,Diamant,E. J., Lowry, S. R., and Von Dippe, P. (1963) Changes in patterns of enzymes of carbohydrate metabolism in the developing rat liver. 7. Biol Chem. 238,2267-2273.

6. Burch H. B., Choi, S., Dence, C. N., Alvey, T. R., Cole, B. R., and Lowry, O. H. (1980) Metabolic of effects of fructose loads in different parts of the rat nephron. 7. Biol Chem. 255,8239-8244.

7. Passonneau, J. V., Lust, D. W., and Crites, S. K. (1977) Studies on the GABAergic system in astrocytoma and neuroblastoma cells in culture. Neurochem. Res. 2, 605-617.

8. Pusateri, M. E., Carter, J. G., Berger, S. J., and Lowry, O. H. (1984) Distribution of three enzymes of GABA metabolism in monkey retina. 7. Neurochem. 42, 1269-1273.

9. Chi, M. M.-Y., Manchester, J. K, Yang, V. C, Curato, A. D., Strickler, R. C, and Lowry, O. H. (1988) Contrast in levels of metabolic enzymes in human and mouse ova. Biol. ofReprod. 39,295-307.

10. Hintz, C. S., Turk, W. R., Cambon, N., Burch, H. B., Nemeth, P. M., and Lowry, O. H. (1985) A method for branched-chain amino acid aminotransfersse activities in microgram and nanogram tissue samples. Anal. Biochem. 146,418-422.

11. Burch, H. B., Cambon, N., and Lowry, O. H. (1985) Branched-chain amino acid aminotransferase along the rabbit and rat nephron. Kidney International 20,114-117.

12. Ichihara, A. and Koyama, A. (1966) Transaminase of branched chain amino acids. I. Branched chain amino acids a-ketoglutarate transaminase. 7. Biochem. (Tokyo) 59,160-169.

13. Taylor, R. T. and Jenkins, W. T. (1966) Leucine aminotransferase. I. Colorimetric assays. 7. Biol. Chem. 2A\, 4391-4395.

14. Lowry, C. V., Kimmey, J. S., Felder, S., Chi, M. M.-Y., Kaiser, K. K., Passonneau, P. N., Kirk, K. A., and Lowry, O. H. (1978) Enzyme patterns in single human muscle fibers. 7. Biol. Chem. 253,8269-8277.

15. Dietrich, W. D., Durham, D., Lowry, O. H., and Woolsey, T. A. (1981) Quantitative histochemical effects of whisker damage on single identified cortical barrels in the adult mouse. 7. Neurosci. 1,929-935.

16. Lowry, O. H., Berger, S. J., Carter, J. G., Chi, M. M.-Y., Manchester, J. K., Knor, J., and Pusateri, M. E. (1983) Diversity of metabolic patterns in human brain tumor enzymes of energy metabolism and related metabolites and cofactors. 7. Neurochem. 41,994-1010.


17. Lowry, O. H., Berger, S. J., Chi, M. M-Y., Carter, J. G., Blackshaw, A., and Outlaw, W. (1977) Diversity of metabolic patterns in human brain tumors. I. High energy phosphate compounds and basic composition. 7. Neurochem. 29, 959-977.

18. Hintz, C S., Lowry, C. V., Kaiser, K., McKee, D., and Lowry, O. H. (1980) Enzyme levels in individuat rat muscle fibers. Am. J. Physiol 239, C58-C65.

19. Bass, A., Brdiczka, D., Eyer, S., Hofer, S., and Pette, D. (1969) Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10,198-206.

20. Burch, H. B., Narins, R. G., Chu, C, FagioU, S., Choi, S., McCarthy, W., and Lowry, O. H. (1978) Distribution along the rat nephron of three enzymes of gluconeogenesis in acidosis and starvation. Am J. Physiol. 235, F246-F253.

21. Chi, M. M.-Y., Hintz, C. S., Henriksson, J., Salmons, S., HeUendahl, R. P., Park, I. L, Nemeth, P. M., and Lowry, O. H. (1986) Chronic stimulation of mammalian muscle: Enzyme changes in individual fibers. Am. J. Physiol 251, C633-C642.

22. Burch, H. B., Bross, T. E., Brooks, C. A., Cole, B. R., and Lowry, O. H. (1984) The distribution of six enzymes of oxidative metabolism along the rat nephron. /. Histochem. Cytochem. 32,731-736.

23. Kato, T. and Lowry, O. H. (1973) Enzymes of energy converting systems in individual mammalian nerve cell bodies. 7. Neurochem. 20,151-163.

24. Lowry, O. H., Roberts, N. R., and Lewis, C. (1956) The quantitive histochemistry of the retina. J. Biol. Chem. 220,879-892.

25. Lowry, O. H., Roberts, N. R., and Chang, M-L. W. (1956) The analysis of single cells. J. Biol. Chem. 222,97-107.

26. Kato, T. and Lowry, O. H. (1973) Distribution of enzymes between nucleus and cytoplasm of single nerve cell bodies. J. Biol. Chem. 248,2044-2048.

27. Curthoys, N. P. and Lowry, O. H. (1973) The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic, and alka-lotic rat kidney. J. Biol. Chem. 248,162-168.

28. Curthoys, N. P. and Kuhlenschmidt, T. (1975) Phosphate-independent glutaminase from rat kidney: partial purification and identity with gamma-glutamyltrans-petidase. J. Biol. Chem. 250,2099-2105.

29. Lowry, O. H., Shulz, D. W., and Passonneau, J. V. (1964) Effects of adenylic acid on kinetics of muscle phosphorylase a. J. Biol. Chem. 253, 8269-8277.

30. Hsieh, B., Chi, M. M.-Y., Knor, J., and Lowry, O. H. (1979) Enzymes of glycogen metabolism and related metabolites in preimplantation mouse embryos. Dev. Biol. 72,342-349.

31. Henry, C. G. and Lowry, O. H. (1985) Enzyme and metabolites of glycogen metabolism in canine cardiac Puikinje fibers. Am. J. Physiol. 248, H599-H605.

32. Passonneau, J. V. and Rottenberg, D. A. (1973) An assessment of methods for measurement of glycogen synthetase activity including a new direct one-step assay. Anal. Biochem. 51,528-541.

33. Berger, S. J., DeVries, G. W., Carter, J. G., Schulz, D. W., Passonneau, P. N., Lowry, O. H., and Ferrendelli, J. A. (1980) The distribution of the components of the cyclic GMP cycle in retina. J. Biol. Chem. 255,3128-3135.

References 387

34. Cole, B. R., Hays, A. E., Boylan, J. B., Burch, H. B., and Lowry, O. H. (1982) Distribution of enzymes of adenylate and guanylate metabolism in rat nephron. Am. J. Physiol 243, F349-355.

35. Teutsch, H. F. and Lowry, O. H. (1982) Sex specific regional differences in hepatic glucokinase activity. Biochem. Biophys. Res. Comm. 106,533-538.

36. Seltzer, J. L. and McDougal, D. B., Jr. (1975) Enzyme levels in chick embryo heart and brain from 1-21 days of development. Develop. Biol. 42,95-105.

37. Burch, H. B., Kuhlman, A. M., Skerjance, J., and Lowry, O. H. (1971) Changes in patterns of enzymes of carbohydrate metabolism in the developing rat kidney. Pediatrics 47,199-296.

38. Holowach, J., Kauffman, F., Beossi, M. G., Thomas, C, and McDougal, D. B. Jr. (1968) The effects of a thiamine antagonist, pyrithiamine, on levels of selected metabolic intermediates and on activities of thiamine-dependent enzymes in brain and liver. JNeurochem. 15,621-631.

39. Mourad, N., and Parks, R. E., Jr. (1966) Erythrocytic nucleoside diphosphokinase II. Isolation and kinetics. J. Biol Chem. 241,271-278.

40. Goffeau, A., Pedersen, P. L., and Lehninger, A. L. (1967) The kinetics and inhibition of the adenosine diphosphate adenosine triphosphate exchange catalyzed by a purified mitochondrial nucleoside dij*osphokinase./.Bw/.C/^m. 242,1845-1853.

41. Lowry, O. H. and Passonneau, J. V. (1966) Kinetic evidence for multiple binding sites on phosphofructokinase. J. Biol. Chem. 241,2268-2279.

42. Kahana, E. E., Lowry, O. H., Schulz, D. W., Passonneau, J. V., and Crawford, E. J. (1961) The kinetics of phosphoglucoisomerase. J. Biol Chem. 235,2178-2184.

43. Lowry, O. H., Roberts, N. R., Schulz, D. W., Clow, J. E., and Clark, J. R. (1961) Quantitative histochemistry of retina. II. Enzymes of glucose metabolism. /. Biol. Chem. 236,2813-2820.

44. Cornell, N. W., Leadbetter, M., and Veech R. L. (1979) /. Biol. Chem. 254, 6627-6527.

45. Lowry, O. H. and Passonneau, J. V. (1964) The relationships between substrates and enzymes of glycolysis in brain. J. Biol. Chem. 239,31-42.

46. Pitts, F. N., Jr. and Quick, C. (1965) Brain succinic semialdehyde dehydrogenase. I. Assay and distribution. J. Neurochem. 12,893-900.

47. Middleton, B. (1973) The oxoacyl-coenzyme A thiolases of animal tissues. Biochem. J. 132,717-730.

48. Corder, C. N., Berger, M. L., and Lowry, O. H. (1974) Quantitative histochemistry of uridine diphosphoglucose-6-pyrophosphatase and uridine diphospho-glucose-pyrophosphorylase in developing rat kidney. J. Histochem. Cytochem. 22,1034-1038.

Chapter 9 1. Linderstrom-Lang, K., Holter, H., and Ohlsen, A. S. (1935) Studies on enzy

matic histochemistry XIII. The distribution of enzymes in the stomach of pigs. Compt.-Rend. Lab. Carlsberg, Serie Chim. 20,66-125.

2. Glick, D. (1961,1963) Quantitive Chemical Techniques ofHisto- and Cytochemistry, vols. 1 and 2 (Wiley [Interscience]), New York.


3. Cole, B. R., Boylan, J. G., Bross, T. E., Burch, H. B., and Lowry, O. H. (1988) Progressive enzyme changes within anatomically defined segments of rat nephron: Demonstration of a new technique. / Histochem Cytochem 36,285-289.

4. Barbehenn, E. K., Wales, R. G., and Lowry, O. H. (1974) The explanation for the blockade of glycolysis in early mouse embryos. Proc. Natl Acad. ScL USA 71,1056-1060.

5. Chi, M. M.-Y., Manchester, J. K., Yang, V. C, Curato, A. D., Strickler, R. C, and Lowry, O. H. (1988) Contrast in levels of metabolic enzymes in human and mouse ova. Biol ofReprod. 39,295-307

6. Outlaw, W. H., Jr. and Lowry, O. H. (1977) Organic acid and potassium accumulation in guard cells during stomatal opening. Proc. Natl. Acad Sci. USA 74,4434-4438.

7. Wenger, B. (1955) Personal communication.. 8. Ess6n, B. Jansson, E., Henriksson, J., Taylor, A. W., and Saltin, B. (1975)

Metabolic characteristics of fibre types in human skeletal muscle. Acta Physiol Scand. 95,153-165.

9. Lowry, C. V., Kimmey, J. S., Felder, S., Chi, M. M.-Y., Kaiser, K. K., Passonneau, P. N., Kirk, K. A., and Lowry, O. H. (1978) Enzyme patterns in single human muscle fibers. /. Biol Chem. 253, 8269-8277.

10. Hintz, C. S., Lowiy, C. V., Kaiser, K. K., McKee, D., and Lowry, O. H. Enzyme levels in individual rat muscle fibers. Am. J. Physiol 239, C58-C65.

Chapter 12 1. Lowry, O. H. (1963) The chemical study of single neurons. Harvey Lect. 58,

1-19. 2. Matschinsky, F. M., Passonneau, J. V., and Lowry, O. H. (1968a) Quantitative

histochemical analysis of glycolytic intermediates and cofactors with an oil well technique. J. Histochem. Cytochem. 16,29-39.

3. Matschinsky, F. M., Rutherford, C. R., and Ellerman, J. E. (1968) Accumulation of citrate in pancreatic islets of obese hyperglycemic mice. Biochem. Biophys. Res. Commun. 33,855-862.

Appendix Good, N. E. (1962) Uncoupling of the Hill reaction from photophosphorylation by

anions. Arch. Biochem. Biophys. 96,653-661. Good, N. E., Winger, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R.

M-M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467-477.

Preface Bergmeyer, H. V. (1970) in Methods of Enzymatic Analysis, 2nd ed. (Verlag Chemie,

Weinheim).

Index

A Acid, definition, 365 Acids, normality of common,

table, 379 ADP, measurement of, 110-114

fluorometric 0.1-8 nmol, 113 kinetics of analytic enzymes, 113 spectrophotometric 15-120

nmol, 112 ADP, effect on glutamate

dehydrogenase, 161,169 removal from ATP, 131

AMP, measurement of, 111-117 catalytic method, with phospho-

rylase a Method II, 114-117 cycling 1-10 fmol, 115 fluorometric direct 50-500 pmol, 115

indirect 4-40 pmol, 115 kinetics of phosphorylasea, 116 precautions in tissue

measurement, 116 spectrophotometric, 114

direct measurement with pyruvate kinase and lactate dehydrogenase Method I, 111-114

fluorometric 0.1-4 nmol, 113 spectrophotometric

15-120 nmol, 112 AMP, effect on phosphorylase a

kinetics, 202, 265

presence in and removal from NADH, 113

presence in NADP*, 116 ATP-ADP cycle, 103-107

conversion of AMP to ATP, 104 cycling reagent for, 104 effect of time, temperature, and

enzyme concentration, 105 indicator reagent for, 104 kinetics of, 107 procedure with NADP cycle

supplement, 1-10 fmol, 106 sample procedure, 1-10 pmol,

105 total adenylate measurement,

2-10 pmol, 106 ATP, measurement with NADPH

as product, 121-126 cycling, 1-10 pmol, 123

0.2-1 pmol, 124 20-100 fmol, 125

fluorometric 0.1-10 nmol, 122

enzyme contamination problems, 122

kinetics of hexokinase, 125 spectrophotometric 15-100

nmol, 121 ATP, removal from ADP, 123 Adenylate deaminase (EC 3.5.4.6),

measurement of enzyme activity, 231-233

389

390 Index

fluorometric, direct 2-8 nmol product, 232

indirect, 0.2-1 nmol product, 232 spectrophotometry, 20-90 nmol

product, 231 Adenylate kinase, contamination in

adenylate measurement, 126

Adenylate kinase (EC 2.7.4.3), measurement of enzyme activity, 233-234

fluorometric, 1-10 nmol product, 234

spectrophotometric, 50-100 nmol product, 233

Adenylates, measurement of with luminescence, 126-128

ATP, 0.1-80 pmol, 126 myokinase, inhibition of by Ap5 A,

127 total adenylate measurement, 128

Alanine, measurement of, 117-118 fluorometric 0.1-8 nmol, 118 kinetics of alanine transaminase,

118 spectrophotometric 15-120

nmol, 117 Alanine transaminase (EC 2.6.1.2),


fluorometer, indirect assay, 0.2-10 nmol product, 235

Aldolase (EC 4.1.2.13), measurement of enzyme activity, 235-237

fluorometric, direct assay, 1-8 nmol product, 236

fluorometric, indirect assay, 0.1-5 nmol product, 236


Alkaline fluorescence of pyridine nucleotides, see specific nucleotide

y-Aminobutyrate transaminase (EC 2.6.1.0)

cycling assay, 5-25 pmol product, 238

fluorometric, 1-10 nmol product, 238


spectrophotometric 30-150 nmol product, 237

y-Aminobutyric acid, measurement of, 154-157

cycling, 1-10 pmol, 156 fluorometric, direct assay,

3-10 pmol, 155 2-step assay, 1-10 pmol, 155 strong alkali method,

1O-100 pmol, 155 kinetics of "Gabase," 156 spectrophotometric, 15-200

nmol, 155 Amytal (amobarbital), inhibitor of

NADH oxidation, 262, 269,281,292,312

Analytical problems and suggestions for remedies, see Chapter 8

Ascorbic acid, as prevention of NADH oxidation, 16,264,291

Aspartate, measurement of, 118-121 cycling, 5-25 pmol, 120

0.1-5 pmol, 120 fluorometric 0.1-8 nmol, 119 kinetics of aspartate

transaminase, 119 spectrophotometric 15-120

nmol, 119

Index 391

Aspartate transaminase (EC 2.6.1.1), measurement of enzyme activity, 239-240

cycling, 10-50 pmol product, 240 fluorometric, direct, 1-10 nmol

product, 240 indirect, 0.1-1 nmol product, 240 spectrophotometric,

20-100 nmol product, 239 Atomic weights, abbreviated list,

380

B Balances, see also quartz-fiber

fishpole balance, 337 balance calibration, 349

colorometric, 349 fluorometric, 349 with frozen-dried tissue, 350

balance case, 344 balance fibers, 342 balance pans, 343 cleaning of, 351 lighting of, 347 linearity of, 349 mounting the fiber, 346

Base/acid ratio for buffers, table, 367, 368

Bases, normality of commonly used, 379

Branched chain amino acid aminotransferase (EC 2.6.1.42) measurement of enzyme activity, 241-242

fluorometric, indirect assay 0.1-1 nmol product, 241

Buffers, definition, 366 how to calculate composition of,

368 table of, 374

Carnitine acetyltransferase (EC 2.3.1.7), measurement of enzyme activity, 242-243

fluorometric indirect assay, 0.5-10 nmol product, 243

Citrate, measurement of, 128-130 fluorometric 1-8 nmol, 129 kinetics of citrate lyase, 129 spectrophotometric 50-120

nmol, 128 Citrate synthase (EC 4.1.3.7),

measurement of enzyme activity , 244-245


fluorometric indirect assay, 0.3-6 nmol product, 244

Conjugate base (or acid), 366 Creatine, meaurement of, 130-132

fluorometric 1-8 nmol, 131 kineticsof creatine kinasereaction,

132 spectrophotometric 15-120 nmol,

130 side reactions of creatine kinase

(ATPase), 131 Creatine phosphate, see P-creatine Creatine phosphokinase (EC 2.7.3.2),


fluorometric, direct assay, 1-10 nmol product, 246

indirect assay, 1-10 nmol product, 247

special requirements for muscle, 247


Cycling, enzymatic, principles of, 85

392 Index

kinetics of, 87 specific first-order rate constant,

82 Cycling, specific procedures,

see specific cycles or assays

D Defatting of frozen dried tissue

samples, 335 2-Deoxyglucose and 2-deoxy-

glucose-6-phosphate, measurement of , 132-134

fluorometric 2-10 nmol total, 134 kinetics of glucose-6-phosphate

dehydrogenase, 132 spectrophotometric 15-100nmol,

133 Design and development of new

methods, 317 P1 ,P5-di(adenosine-5')penta-

phosphate, adenylate kinase inhibitor, 117,127

Dihydroxyacetone phosphate, measurement of with

glycero-P dehydrogenase, Method 1,134-138

cycling assay, 0.5-10pmol, 136 0.1-.5pmol, 137

fluorometric assay 1-8 nmol, 135 indirect 10-100 pmol, 136

kinetics of glycero-P dehydrogenase, 135,137

special precautions, 137 measurement of with glyceralde-

hyde 3-P dehydrogenase Method II, see Fructose-1,6-bisphosphate Method II

Dissection of frozen-dried tissue sections, 329

histological identification, 334

Dissociation constant, definition for acid or base, 366

of acid andbases for buffers, table, 374

of water, 369 Dithiothreitol, to reduce glutathione,

139,141,162,202,314 Dry loading of tissue sections

for analysis, 354 Drying of frozen tissue under

vacuum, 326

E Enzyme activities, measurement of,

see specific enzyme, 229-305 Enzyme activity, unit of, 229 Enzyme stability at high dilution,

353 Evacuation of frozen-dried tissue

sections, 329

F Fluorescence of pyridine nucleotides,

4,5 conversion by alkali to fluorescent

products, 10 effect of enzymes on, 6 effect of pH on, 6 enhancement of, 6,11 filters for measurement, 9-10 increased fluorescence in alkali,

12-13 interfering substances, 12 quenching of, 7

Huorometry fluorometers, 20-21 pyridine nucleotides, useful range, 5 quenching, 5 reference standards, 8

Freezing of tissues for histochemistry, 322 for metabolites, 72-73

Index 393

Fructose, measurement of, 138-139 fluorometric 0.1-10 nmol, 139 kinetics of hexokinase for fructose,


138 Fructose-1,6-bisphosphatase

(EC 3.1.3.11) measurement of enzyme activity, 248-250


fluorometric direct assay, 5-10 nmol product, 249

indirect assay, 5-10 nmol product, 249

kinetics of enzyme, 250 Fructose-1,6-bisphosphate,

measurement of with aldolase and glycero-P

dehydrogenase Method I, 147-148

fluorometric 0.05-4 nmol, 147 kinetics of auxiliary enzymes,


147 aldolase and glyceraldehyde-P

dehydrogenase Method II, 148-152

cycling, 2-20 pmol, 150 0.1-1 pmol, 151

fluorometric 0.1-10 nmol, 150 kinetics of auxiliary enzymes

151 spectrophotometric

8-100 nmol, 149 Fructose-6-phosphate, measurement

of with glucose-6-P dehydrogenase

and P-glucoisomerase Method 1,140-141

fluorometric 0.1-10 nmol, 141 kinetics of P-glucoisomerase

141 spectrophotometric 15-200

nmol, 140 P-fructokinase,Methodn, 142-144

cycling, 10-40 ftnol, 143 fluorometric 0.1-5 nmol, 143 spectrophotometric

8-100 nmol, 142 Fructose-6-phosphate and mannose-

6-phosphate measurement of, 144-146

cycling, 0.1-1 pmol, 145 fluorometric 0.1-10 nmol, 145 kinetics of P-glucoisomerase, 146 kinetics of P-mannoisomerase,


146 Fumarase(EC3.1.3.1 l)measurement

of enzyme activity, 250-252

cycling, 50-150 pmol product, 251

fluorometric, indirect assay, 3-10 nmol product, 251


Fumarate, meaurement of, 152-154 fluorometric, direct assay

0.1-10 nmol, 153 fluorometric, 2-step assay, 153 kinetics of fumarase, 154 spectrophotometric 15-200 nmol,

152

GTP-GDP cycle, 107-110 comments on cycle, 109 cycling reagent for, 108

394 Index

effects of time, temperature, and enzyme concentration, Table 9,108

indicator reagent for, 109 Glucose, measurement of, 157-160

cycling, 1-10 pmol, 158 10-50 fmol, 159

destruction with alkali, 177 fluorometric, 0.1-10 nmol, 157 kinetics of hexokinase, 159 spectrophotometric, 15-200nmol,

159 Glucose-1-P, measurement of,

160-162 fluorometric, 0.1-10 nmol, 161 kinetics of P-glucomutase, 161 spectrophotometric, 15-200nmol,

160 Glucose-1,6-bisphosphate,

measurement of, 165-167 fluorometric, 2-8 pmol, 166 kinetics of P-glucomutase, 166 modification for 10 fmol, 166

Glucose-6-phosphatase (EC 3.1.3.9) measurement of enzyme activity, 252-253

cycling, 10-100 pmol product, 253 fluorometric indirect assay,

1-10 nmol product, 253 spectrophotometric, 20-100 nmol

product, 253 tissue homogenate preparation for

assay, 252 Glucose-6-phosphate, measurement

of, 162-164 cycling, 1-10 pmol, 162

0.1-1 pmol, 164 10-50 fmol, 164

fluorometric, 0.1-10 nmol, 162 kinetics of glucose-6-P

dehydrogenase, 164

spectrophotometric, 15-200nmol, 162

Glucose-6-P dehydrogenase (EC 1.1.1.49),


cycling, 5-25 pmol product, 255 fluorometric direct assay,

1-10 nmol product, 255 spectrophotometric, 50-150nmol

product, 255 Glutamate, measurement of, 167-170

cycling, 2-10 pmol, 168 0.5-2.5 pmol, 168

fluorometric, 0.1-10 nmol, 168 kinetics of glutamate

dehydrogenase, 169 spectrophotometric, 15-200nmol,

167 Glutamate decarboxylase

(EC 4.1.1.15) measurement of enzyme activity,

256-258 cycling, 0.3-1.2 nmol product, 257 fluorometric indirect, 1.8-10pmol

product, 257 spectrophotometric, 30-100 nmol

product, 257 Glutamate dehydrogenase


258-259 cycling, 3-6 pmol product, 259 fluorometric indirect, 1-3 nmol

product, 259 spectrophotometric, 25-150 nmol

product, 258 Glutaminase (EC 3.5.1.2)



Index 395

fluorometric indirect, 1-10 nmol product, 261

spectrophotometry, 25-100 nmol product, 260

Glutamine, measurement of, 170-171 cycling, 0.2-1.5 pmol, 171 fluorometric, 1-10 nmol, 170

Glycerol, measurement of, 171-172 fluorometric, 0.1-10 nmol, 172 spectrophotometry 15-200

nmol, 172 oc-Glycerophosphate, measurement of

Methodlwitha-glycero-Pdehydro-genaseandhydrazine, 172-174

fluorometric, 0.2-10nmol, 173 kinetics of reaction, 173 spectrophotometry,

15-200 nmol, 173 Method II with triose-P isomerase

and glyceraldehyde-3-P dehydrogenase, 174-176

fluorometric direct 0.1-5 nmol, 175

kinetics of glyceraldehyde-3-P dehydrogenase, 176

spectrophotometric, 8-100 nmol, 175

oc-Glycero-3-P dehydrogenase (EC 1.1.1.8)


cycling, 10-100 pmol product, 264 modification for muscle tissue, 264

fluorometric, direct 2-8 nmol product, 262 indirect 0.1-3 nmol product, 263


Glycogen, measurement of, 177-179 cycling, l-10pmolglucosyl units,

178 fluorometric direct 0.1-10

glucosyl units, 178 spectrophotometric 15-200 nmol

glucosyl units, 177 Glycogen phosphorylase (EC 2.4.1.1)


cycling 10-50 pmol product, 266 modification for muscle tissue, 267

fluorometric direct 1-10 nmol product, 266 indirect 1-5 nmol product, 266


tissuepreparationforaandhforms, 265

Glycogen synthase (EC 2.4.1.11), measurement of enzyme activity, 268-270


fluorometric indirect, 1-8 nmol product, 269

tissue preparation for i anddforms, 268

Guanine nucleotides, measurement of, 179-183

cycling, 1-3 pmol, 182 fluorometric 0.1-10 nmol,

180 kinetics of succinyl Co A

synthetase and pyruvate kinase, 181

removal of ADP as a contaminant, 181

396 Index

Guanine phophoribosyl transferase, see hypoxanthine phosphoribosyltransferase

Guanylate kinase (EC 2.7.4.8) measurement of enzyme activity, 270-272

cycling 0.3-2 pmol product, 271

fluorometric 2.5-5 nmol product, 271

spectrophotometry 25-100 nmol product, 270

H Hair-points for frozen-dried tissue

sections, 333 Henderson-Hasselbalch equation,

366 Hexokinase (EC 2.7.1.1)


cycling 1-10 pmol product, 273 fluorometric direct 1.5-10 nmol

product, 273 indirect 1-10 nmol product, 273 spectrophotometric 50-150 nmol

product, 173 Hydrazine as trapping agent for

oc-keto acid, see assays for P-pyruvate, lactate, glycerol, a-glycero-P, malate

p-Hydroxyacyl coenzyme A dehydrogenase (EC 2.4.2.8)


cycling 20-100 pmol product, 275 fluorometric indirect 0.1-3 nmol

product, 275 spectrophotometric 20-150 nmol

product, 274

Hypoxanthine (guanine) phosphoribosyl tranferase (EC 2.4.2.8) measurement of enzyme activity, 276-277

fluorometric indirect 0.5-5 nmol product, 276

I International unit of enzyme activity,

229 Isocitrate, measurement of, 183-184


kinetics of isocitrate dehydrogenase, 184

spectrophotometric 15-200 nmol, 183

Isocitrate dehydrogenase (NAD) (EC 1.1.1.41)

meaurement of enzyme activity, 277-278

fluorometric 1-10 nmol product, 277

Isocitrate dehydrogenase (NADP) (EC 1.1.1.42)


cycling 2-10pmol product, 278 fluorometricindirect5-20nmol

product, 278

K Katal, unit of enzyme activity, 229 Ketoacid CoA transferase


279-280 cycling, 2-25 pmol product,

280 fluorometricindirect2-10nmol

product, 280

Index 397

a-Ketoglutarate, measurement of, 185-188

cycling, 1-10 pmol, 186 equilibrium of glutamate

dehydrogenase reaction, 186 fluorometric direct 2-8 nmol, 185

indirect 0.2-1 nmol, 185 kinetics of reaction, 187 spectrophotometric 15-200nmol,

185 a-Ketoglutarate dehydrogenase


280-281 fluorometric direct 1.5-10 nmol

product, 281 Kinetics, enzyme

factors affecting, 48-49 first order, 29-32 first order rate constant, 24, 33 half-time, definition, 24-25 Michaelis-Menten equation, 31 mixed zero-order and first order,

33-35 one-step reaction, one substrate,

29-32 one-step reaction, two substrates,

35-41 "random" order of addition,

37-38 "cooperative addition," 38-41 "ordered addition," 41

reversible first-order, 25 relaxation time, 25 second order rate constant, 38 second order reactions, 41-43 two-step reactions, 43-48

both steps first order, 45 first step zero order, second step

first order, 43 lag time, 45

Kinetics, nonenzyme, 23-28 first order reactions, 24-27 order of reaction, 23 second order reactions, 27-28

Kinetics of enzymes for assays of metabolites and enzyme actvities, see specific assay

Knives for frozen-dried tissue microdissection, 330-332

L Lactate, measurement of

with glutamate-pyruvate transaminase, Method 1,188-193

contamination problems, 192 cycling 1-10 pmol, 191

0.1-1 pmol, 191 2O-100 fmol, 191

fluorometric direct 0.2-10 nmol, 190 indirect 0.1-10 nmol, 190

kinetics of glutamate-pyruvate transaminase, 193

kinetics oflactatedehydrogenase (beef heart), 192

kinetics of overall reaction, 192 spectrophotometric 15-200

nmol, 189 withhydrazine, Method n, 193-195

fluorometric 0.5-10 nmol, 194 spectrophotometric 15-200

nmol, 194 Lactate dehydrogenase (EC 1.1.1.27)

measurement of enzyme activity

Method I cycling 50-500 pmol product, 282



398 Index

Method II fluorometricindirect2-10nmol

product, 283 Log table, homemade, 371 Logarithms (to base 10), 365

M Malate, measurement of

Method I with aspartate transaminase, 195-197

fluorometric 0.1-10 nmol, 196 kineticsofmalatedehydrogenase

and aspartate transaminase, 196


MethodH withhydrazine, 198-199 fluorometric 0.1-10 pmol, 198 spectrophotometric 15-200

nmol, 198 Malate dehydrogenase (EC 1.1.1.37)

measurement of enzyme activity

Method I cycling 5-250 pmol product, 285 fluorometric direct 2-10 nmol

product, 284 indirect 0.2-2 nmol product, 284


Method II fluorometricindirect2-10nmol

product, 286 Malate dehydrogenase

(decarboxylating) (BC1.1.1.40)measurementof enzyme activity, 287

cycling 2 pmol product, 287 fluorometric 5-10 pmol product,

287

Michaelis constant, 31 Modification of methods

enzyme measurement methods, 309-312

metabolite measurement methods, 312

Mounting tissues for sectioning, 323

N NAD cycle, 96-97

blank problems, 101-103 cycling reagent for, 97 effects of pH and temperature

on kinetics, 100 effects of time, temperature and

enzyme concentration, Table 7,99

indicator reagent for, 98 kinetic of cycling enzymes, 100 maximum cycling rate, 101 modification of, 98 removal of NAD from enzymes, 97 sample procedure, 0.2-2 pmol, 98

10-100 fmol, 99 NAD+

conversion to alkaline fluorescent product, 10

stabilization with imidazole, 11 destruction with weak alkali, 16

effect of temperature, 17 preparation of solutions, 19

standardizations, 19 stability in acid, 16

NADH conversion to alkaline fluorescent

product, 12 stabilization with imidazole, 11

degradation product inhibitory to enzymes, 16

destruction in acid, 3,13-14 fluorescence of, 4

Index 399

enhancement of fluoresence by enzymes-oc-glycero-P dehydrogenase, 147,173,176 lactate dehydrogenase, 118,120 malate dehydrogenase, 119

oxidation in small volumes, 16 preparation of solutions, 19

standardization of, 20 spectral absorption, 4 stability in storage, 15

NADP, cycle I, 88-96 double cycling, 94

sample procedure, 0.2-2 fmol, 95

effect of time, temperature, enzyme activity, 90-91

heat inactivation, precaution, 91-92

indicator reagent for, 90 reagent, 89-90 sampleprocedure, 0.1-1 pmole,91

0.01-.lpmol,93 sensitivity extension by reduction

in volume, 93 spectrophotometric procedure,

92 table for cycling rates, 89 useful cycling conditions for

fluorometric procedures fluorometric in 0.1 mL,

Table 2,91 spectrophotometric in 0.1 mL,

Table 3,93 fluorometric in 10 ^L, Table 4,

94 fluorometric in 2 |iL, Table 5,

94 NADP, cycle II, 96

yield with useful cycling procedures, Table 6,96

NADP+ conversion to alkaline

fluorescent product, 10 stabilization withimidazole, 11

destruction with alkali, 3,16 as an analytical step, 17 effect of temperature, 17

preparation of solutions, 19 standardization of, 19

NADPH conversion to alkaline fluorescent

product, 12 stabilization withimidazole, 11

destruction in acid, 3,13-14 fluorescence of, 4 spectral absorption, 4 stability in storage, 15 enhancement of, 6

Nicotinamide, inhibitor of NAD+

destruction, 237,264, 311

Nucleoside diphosphokinase (EC 2.7.4.6)



Nucleotide triphosphates, see Total nucleotide triphosphates

O Oil well technique, 355-362

addition of sample/reagent, 360-361

cleaning of oil well racks,357 composition of racks, 355 heating and cooling of racks,

357 microscope and stage, 358 oil composition, 356 oil well pipets, 52, 61, 358

400 Index

Oxaloacetate, measurement of 199-200

fluorometric 0.1-8 nmol, 199 kinetics of malate dehydrogenase,


199 Oxaloacetate, stability of,

97,199, 284

pH of free acids and bases, 369 pK, see dissociation constant Peroxide, hydrogen

conversionofpyridine nucleotides to fluorescent product,

seeNADH,NADPH destructionof a-keto acids, 112,169,

238,254, 269,290, 377 oxidation of NADH, 167

Phosphate, measurementpf, 200-203 cycling, 1-10 pmol, 201 fluorometric 1-10 nmol, 201

indirect, 1-10 nmol, 201 kinetics of phosphorylase a,

202-203 kinetics of overall reaction, 203 spectrophotometric 15-200nmol,

200 Phosphocreatine

measurement with pyridine nucleotides, 121-126

cycling 1-10 pmol, 123 cycling 0.2-1 pmol, 124 cycling 20-100 fmol, 125 fluorometric, 122 kinetics of creatine kinase,

126 spectrophotometric, 121

measurement with luminescence, 0.1-80 pmol, 126

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32)


cycling 5-50 pmol product, 291 fluorometric indirect 2-8 nmol

product, 290 Phosphofructokinase (EC 2.7.1.11)


fluorometric direct 1-10 nmol product, 293 indirect 0.1-5 nmol product, 293


Phosphoglucoisomerase (EC 5.3.1.9) measurement of enzyme activity,

292-294 fluorometric direct 2-10 nmol

product, 294 indirect 100-500 pmol product, 294


Phosphoglucomutase (EC 2.7.5.1) measurement of enzyme

activity, 294-296 fluorometric direct 2-10 nmol

product, 295 indirect 3-10 nmol product, 295 indirect 0.2-2 nmol product, 296


6-Phosphogluconate, measurement of, 203-205


kinetics of 6-phosphogluconate

Index 401

dehydrogenase, 204 spectrophotometric 15-200 nmol,

203 6-Phosphogluconate dehydrogenase


296-297 cycling 10-50 pmol product, 296 fluorometric 1-10 nmol

product, 297 spectrophotometric 50-150

nmol product, 297 3-Phosphoglycerate, measurement of

Method I by NADH oxidation, 205-207

fluorometric 0.2-8 nmol, 206 kinetics of P-glycerate kinase,

206 kinetics of glyceraldehyde-3-P

dehydrogenase, 206 spectrophotometric 15-200

nmol, 205 Method II indirect by NADH

formation, 207-209 fluorometric 0.1-10 nmol, 208

3-Pho^*K)glyca-atekinase(EC2.7.1.3.) measurement of enzyme activity,

298-299 fluorometric 5-10 nmol

product, 298 spectrophotometric 100-200

nmol product, 298 Phosphorylase, see Glycogen

phosphorylase P-pyruvate, measurement of, 209-211

fluorometric 0.1-8 nmol, 210 kinetics of pyruvate kinase, 211 spectrophotometric 15-200nmol,

209 trapping of preformed pyruvate

with hydrazine, 210

Pipeting errors in, 56 role of surface tension, 51-54 small pipets and tubes, 55 technique, 54-55

Pipets calibration of, 62-66

colormetric, 63-65 gravimetric, 63 volumetric, 65-66

cleaning of, 66-68 construction of, 57-62

glass, 58-61 quartz, 61

oil well, 61 properties of, 57-58 racks for, 68-69 rinsing, 56-57 smaller pipets, 61-62 storage of, 68

Potassium, measurement of, 211-215

blank problems, potassium contamination, 214

cycling 10-100 pmol, 213 1-10 pmol, 213

fluorometric direct 10-100 nmol, 212

indirect 1-10 nmol, 212 for tissue sections 0.2-2 nmol,

212 Problems in enzyme activity

analysis, 316-317 Problems in metabolite analysis,

314-315 Pyrophosphate, measurement of

Method I with UDPG pyrophosphorylase, 215-217

cycling 0.05-0.25 pmol, 217 fluorometric 0.1-9 nmol, 216

402

kinetics of UDPG pyrophosphorylase, 217

spectrophotometry 15-200nmol, 216

Method II with PPrdependent fructose-6-P-kinase,218-219

fluorometric 0.1-4 nmol, 218 spectrophotometry 15-120nmol,

218 Pyruvate, measurement of, 219-222

cycling 1-5 pmol, 221 0.1-.5pmol,221


indirect 10-100 pmol, 220 kinetics of beef heart lactate

dehydrogenase, 222 spectrophotometry 15-120 nmol,

222 Pyruvate kinase (EC 2.7.1.40)


activation by fructose-1,6-bisphosphate, 300

fluorometric direct 1-5 nmol product, 300

indirect 0.1-5 nmol product, 300


Q Quartz fiber fishpole balance,

see Chapter 11 Quartz fibers, selection of for

balances, 339 sensitivity of, formula, 339

Quartz pipets, see Kpets Quinine for fluorometry standards,

8 reference solutions, 8

Index

S Sample carriers, for frozen-dried

tissue, 334 Sample volume, determination of, 336 Schiffs base, formed by

glyceraldehyde-3-P, 149 Sectioning of frozen tissues, 325 Sensitivity, methods to increase

analytical, 313 Simplification of assays, 307

elimination of steps, 307 reduction in scale, 308

Static, control by radioactive source 330,333,335,347,348,359

Statistical shortcuts, 371-373 standard error of the mean, 372 standard deviation, 371

Stock solutions, preparation and storage, table, 375-378

Succinate semialdehyde dehydrogenase (EC 1.2.1.24)


cycling 10-100 pmol product, 301


Sucrose, measurement of, 223-225 cycling 1-10 pmol, 224

0.1-1 pmol, 224 10-100 fmol, 225

fluorometric direct 0.1-5 nmol, 223 indirect 0.01-1 nmol, 224


T Thiolase (EC 2.3.1.9) measurement

of enzyme activity, 302-303

Index 403

cycling 15-60 pmol product, 303 fluorometric indirect 2-5 nmol

product, 302 Tissue extracts, fluorescence of,

77-78 charcoal treatment of, 78 Horosil treatment of, 78 quenching of fluorescence,

77,135,210 removal of fluorescence, 77-78

Tissue, freezing of, see freezing Tissue preparation

extracts for metabolite measurement, 74-78

methanol-acid extracts, 76 neutralization of, 75-76 perchloric acid extract, 74-76

extracts for pyridine nucleotide measurement, 78-81

effect of hemoglobin on, 78-79 oxidized pyridine nucleotides,

79 reduced pyridine nucleotide, 79 total pyridine nucleotides, 79

homogenates for enzyme measurements, 71-72

storage of frozen tissue, 73-74 Tissue sections, 321-327

freezing, 322 mammalian ova, special

preparation, 328 storage of sections, 327 mounting of tissues for section,

323 Total nucleotide triphosphates,

measurement of, 225-227 fluorometric 0.05-4 nmol, 226 kinetic of P-fructokinase, 227 spectrophotometric 8-60 nmol,

226 Troubleshooting

in metabolite assays, 313 blanks, 315 mixing, 315

in enzyme assays, 316 specificity, 316 proportionality, 316

T\ibe racks, 68-69

U Unit of enzyme activity, see

International enzyme unit Uridine 5'diphosphoglucose,

measurement of, 227-228 fluorometric direct 0.05-5 nmol,

227 kinetics of uridine diphosphoglu-

cose dehydrogenase, 228 spectrophotometric 8-100 nmol,

227 Uridinediphosphoglucose

pyrophosphorylase (EC 2.7.7.9)


cycling 15-50 pmol product, 305


W Weighing tissue sections, 347

coirectionforabsorptionof gas and moisture, 349

Documents

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