Protein Measurement with the Folin Phenol Reagent
01 Nov 1951-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 193, Iss: 1, pp 265-275
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
Abstract: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
TL;DR: This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique and demonstrates a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts.
Abstract: Cu + produced during the reaction of protein with alkaline Cu ++ can be monitored by measuring the absorbance at 562 nm of the intense purple complex formed with the ion of bicinchoninic acid (BCA). The color produced is stable and increases in a linear fashion over a broad working range of increasing protein concentration. Since BCA is stable, it is incorporated in the reagent formulation at the start of the reaction. Thus, the method offers mechanical simplification over the method described by Lowry et al.
TL;DR: The purification of homogeneous glutathione S-transferases B and C from rat liver is described, and only transferases A and C are immunologically related.
Abstract: The purification of homogeneous glutathione S-transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2-dichloro-4-nitrobenzene. Although each of the four enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p-nitrobenzyl chloride. Assay conditions for a variety of substrates are included. All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite the similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
TL;DR: The present paper gives a detailed account of the investigations on rabbit liver microsomes and crude microsomal digests, which have led to postulate the hemoprotein nature of the pigment.
Abstract: The presence in mammalian liver microsomes of a carbon monoxide-binding pigment has been reported by Klingenberg (1) and by Garfinkel (2). The CO compound of the reduced pigment has an intense absorption band at 450 rnl.c and thus can be readily detected in dithionite-treated microsomes by difference spectrophotometry. The CO difference spectrum of reduced microsomes is, however, unusual in that it shows no peaks other than that at 450 rnp and, therefore, provides no clue to the nature of the pigment. The elucidation of its nature has further been hampered by the reported lability of the microsomal pigment to detergents, low pH, and enzymatic digestion (1,2). In addition, the CO compound has been reported as not photodissociable (1). In preliminary communications (3, 4), we have reported evidence for the hemoprotein nature of the microsomal CObinding pigment, provisionally called “P-450,” and shown that it can be converted into a solubilised form, which we term “P420,” by treatment of microsomes anaerobically with snake venom or deosycholate. The solubiliaation is accompanied by an unusual change in the spectral properties of the pigment. Further, the solubilized pigment has been partly purified, free from cytochrome 55, and shown to possess absorption spectra characteristic of hemoproteins (5). The present paper gives a detailed account of the investigations on rabbit liver microsomes and crude microsomal digests, which have led us to postulate the hemoprotein nature of the pigment. Purification and properties of the solubilized hemoprotein will be reported in the accompanying paper (6).
TL;DR: The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated.
Abstract: A simple and rapid method is presented for the preparation of I/sup 131/- labeled human growth hormone of high specific radioactivity (240-300 mu C/ mu g). Low amounts of carrierfree I/sup 131/ iodide (2 mC) are allowed to react, without prior treatment, with small quantities of protein (5 mu g) in a highyield reaction (approx. 70% transfer of I/sup 131/ to protein). The degree of chemical substitution is minimized (0.5- 1.0 atom of iodine/molecule of protein) by the use of carrier-free I/sup 131/ iodide. The I/sup 131/-labeled hormone (up to 300 mu C/ mu g) contains no detectable degradation products and is immunologically identical with the unlabeled hormone. The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated. (auth)
TL;DR: A rapid method has been devised which requires only 5 c.mm.
Abstract: The alkaline phosphatase of the serum increases early and markedly in rickets and returns completely to normal only after healing is complete. Because of this fact, serum phosphatase is the most satisfactory index now known for the detection of this deficiency. The phosphatase activity of serum is not strictly specific in this respect and has also proved clinically useful in a number of other pathological states; e.g., Paget’s disease, hyperparathyroidism, liver disease, etc. In connection wit.h nutritional studies on large groups of population, it became necessary to have a rapid method for the determination of this enzyme on small amounts of serum. By the use of a new substrate (pnitrophenyl phosphate) a method has been devised which requires only 5 c.mm. of serum (0.005 ml.) and which permits 50 to 100 analyses to be made in 2 hours. The simplicity and speed of the method recommend it for macroas well as microdeterminations and for either alkaline or acid phosphatase. A number of methods have been described for the determination of the phosphatase content of serum and other biological materials, all of which depend upon the principle of measuring the rate of hydrolysis of various phosphate esters under specified conditions of temperature and pH. The two most widely used methods are those of Bodansky (1) and King and Armstrong (2) in which glycerol phosphate and phenyl phosphate respectively are employed as substrates. While these methods are satisfactory for many uses, they are rather time-consuming when large numbers of determinations are needed; furthermore, they require larger samples of serum than is convenient for the purpose of dietary surveys. The substrate, p-nitrophenyl phosphate, was studied by King and Delory (3) and has been used for phosphatase estimations by Ohmori (4) and by Fujita (5). The compound is colorless, but upon splitting off the phosphate group, the yellow salt of p-nitrophenol is liberated (absorption maximum, 400 mp). Hence the substrate is itself an indicator of the amount of splitting and thus a measure of phosphatase activity. It is only necessary to incubate serum with the buffered reagent, stop the reaction
TL;DR: In this article, the authors summarized research into the existing methods for the quantitative determination of tyrosine and tryptophane in proteins, including the Folin-Looney method, which is based on reaction of a phosphotungstic phosphomolybdic acid in a phenol solution.
Abstract: The article summarizes research into the existing methods for the quantitative determination of tyrosine and tryptophane in proteins. Limitations to the accuracy of the Folin-Looney method (reaction of a phosphotungstic phosphomolybdic acid in a phenol solution, evaluated using colorimetry) have been solved by an improved method detailed in the text. The hydrolysis of proteinaceous material to allow chemical analysis of tryptophane has also been improved; the method is based on digestion with sodium hydroxide for 18-20 hours, followed by rapid neutralization and acidification with sulfuric acid. A more accurate test for tyrosine based on Millon's reaction has been developed; acidified mercuric sulfate solution is used to dissolve precipitated tyrosine and sodium nitrite is added to produce the colored product which is assessed colorimetrically. Two types of casein analyzed by these methods contained 1.4% tryptophane and 6.37-6.55% tyrosine. Tryptophane and tyrosine content of various materials were: casein 1.4%, 6.4-6.6%; egg albumin 1.3%, 4.0%; edestin 1.5%, 4.5%; gliadin 0.84%, 3.1%; zein 0.17%, 5.9%. A method for preparation of the pure mercuric sulfate reagent is described.