Roger L. Lundblad

Colorimetric protein assay techniques

There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G‐250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory‐to‐laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro‐Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody‐based methods. The key point is that whatever method is adopted as the ‘gold standard’ for a given protein, this method needs to be used routinely for calibration.

Thrombogenic Materials in Prothrombin Complex Concentrates

Abstract Prothrombin complex concentrates are now available for use for treatment of bleeding complications associated with deficiencies of factors II, VII, IX, or X. The purpose of this report is ...

Linkage between blood coagulation and inflammation : stimulation of neutrophil tissue kallikrein by thrombin

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Students paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.

Inhibition of the heparin-antithrombin III/thrombin reaction by active site blocked-thrombin☆

Abstract Active site blocked-thrombin, prepared by reacting thrombin with valyl-isoleucyl-prolyl-arginine chloromethyl ketone, inhibits the heparin enhanced-antithrombin III/thrombin reaction. Since active site blocked-thrombin does not interact with antithrombin III it was concluded that active site blocked-thrombin was competing for heparin in the reaction system. The heparin concentration dependence for maximum enhancement of the antithrombin III/thrombin reaction in the presence and absence of active site blocked-thrombin indicated that heparin was binding to thrombin to enhance the reaction rate. A dissociation constant value of 6.4×10−9M was estimated for the heparin·thrombin complex which is similar to the value of 5.8×10−9M previously reported (Griffith M.J. (1979) J. Biol. Chem. in press). Antithrombin III·thrombin complexes were also found to bind heparin with an affinity equivalent to thrombin. The results were interpreted to indicate that heparin binds to thrombin as the first step in the mechanism of action of heparin in enhancing the antithrombin III/thrombin reaction.

Fractionation of heparin by affinity chromatography on covalently-bound human α-thrombin☆

Abstract Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine- p -nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.

Review of methods for determination of total protein and peptide concentration in biological samples

Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit‐for‐use, quality‐by‐design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

Glycosylation in Pichia pastoris.

The Pichia pastoris system for expression of heterologous recombinant proteins is being used increasingly because of the large yields of properly folded proteins that result and the ease of scaling preparations into large-biomass fermentors. Another advantage of this system centres on the type of glycosylation that results, generally yielding protein-bound oligosaccharides that are of much shorter chain length than found in Saccharomyces cerevisiae. This review is a summary of the current state of knowledge of glycosylation of proteins in this methylotrophic yeast.

Approach to assay validation for the development of biopharmaceuticals.

The development, manufacture and characterization of biopharmaceuticals requires the use of a variety of analytical procedures. Each assay must be validated to demonstrate that it is suitable for its intended purpose, either as an in‐process analysis for the characterization of critical product/process attributes or final product characteristics. This paper is a brief review of the various considerations required for the validation of an assay procedure for use within Good Laboratory Practices (‘GLP’) or current Good Manufacturing Practices (‘cGMP’).

The effect of ethionine intoxication on the membrane proteins of rat parotid gland secretory granules

Abstract The effect of chronic ethionine intoxication on the secretory granules of the parotid gland in rats has been investigated. Young adult (160–180 gm) Sprague-Dawley rats were placed on a protein-free diet and given six daily injections of 2% ethionine in saline equivalent to 0.5 gm ethionine/gm body weight/day. On day 7, the survivors were sacrificed and the parotid gland excised. The secretory granules were isolated; granules from both normal and experimental animals were disrupted by osmotic lysis and the membrane and granule contents obtained by centrifugation. Amino acid analysis of the respective membrane preparations showed little, if any, difference between the normal and the ethionine-treated animals. Analysis of the granule contents of DNAse activity showed again little difference between the control and experimental animals. Analysis of the membrane proteins by discontinuous polyacrylamide gel electrophoresis showed small differences in the ethionine-treated animals when compared to control membrane preparations. When these gels were stained for carbohydrate, it was observed that a major membrane glycoprotein was missing in the granule membranes obtained from the ethionine-treated animals. These results suggest that the abnormal secretory response observed in the ethionine-treated animals is a reflection of an alteration in membrane biosynthesis.

Recent articles of interest to biotechnology in other Portland Press journals

The manufacture of protein biotherapeutics via recombinant DNA technology generally involves the expression of a ‘human’ protein in a ‘ foreign ’ cell [e.g. Chinese hamster ovary (CHO) cells, baby-hamster kidney (BHK) cells, yeast cells, bacterial cells]. Correct folding of these proteins depends upon the success of a variety of co-translational and post translational modifications, including the formation of the correct disulphide bonds. Disulphide bonds are required for the maintenance of the correct protein conformation. The protein disulphide isomerase (PDI) family is involved in the processing of these secretory proteins in the endoplasmic reticulum. Ferrari and So