ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 220, No. 2, February 1, pp. 572-575, 1983 Effect of Carbohydrate on Protein Solubility ERLINDA Q. LAWSON,* BO. E. HEDLUND,? MARNA E. ERICSON,* DEBORAH A. MOOD,* GARY W. LITMAN,§ AND RUSSELL MIDDAUGH”’ *Department of Biochemistry, University of Wyoming, Luramie, Wyoming 82071; Departments of tPediatrics and $Laberatwy Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455; and the &%an-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York 10580 Received July 2, 1982, and in revised form October 11, 1982 The effect of covalently attached carbohydrate on the solubility of a number of proteins has been examined by the PEG precipitation technique. Both increases and decreases in solubility are observed depending on the state of glycosylation, the type of protein, and temperature. It is concluded from this data and associated apparent thermodynamic parameters that a general role for carbohydrate in the solubilization of proteins is not currently experimentally supportable. The role of carbohydrate in the struc- ture and function of glycoproteins is un- clear. Covalently attached carbohydrate has been suggested to provide a signal for both protein transport and metabolism as well as serve a general structural role especially with regard to protein stability (1, 2). Another function frequently pos- tulated to be of general utility is an en- hancing effect of carbohydrate on protein solubility (e.g., 3, 4). This idea follows im- mediately from the highly polar nature of carbohydrate moieties. Such a role may also be of some importance in certain dis- ease states such as those involving cryoim- munoglobulins (5) and lens crystallins (6) where changes in protein solubility as an immediate consequence of abnormal gly- cosylation may be implicated in patholog- ical processes. In this communication we employ the recently introduced polyeth- ylene glycol (PEG)’ precipita,tion tech- nique (7-9) to assess the role of carbohy- drate on protein solubility. This question 1 Author to whom all correspondence should he ad- dressed: Department of Biochemistry, University of Wyoming, Box 3944 University Station, Laramie, Wyo. 82071. ’ Abbreviation used: PEG, polyethylene glycol. is addressed by a systematic comparison of the solubility of a number of glycopro- teins with certain of their deglycosylated analogs. MATERIALS AND METHODS Rihonuclease A and B were obtained from Sigma (Type XII-A and XII-B, respectively) and used with- out further purification. Solutions of rihonuclease were heated at 65°C for 10 min to remove aggregates (10). Hemoglobins A,, and Ai. were isolated from di- abetic individuals as described by Tisel et al. (11) and then purified by the method of McDonald et al. (12). Analysis of the Ai, fraction by isoelectric focusing indicated a nearly pure (-95%) fraction. Both prep- arations were converted to ferric hemoglobin using 4 mol of potassium ferricyanide per mole of ferrous heme followed by dialysis. Purification of monoclonal immunoglohulins (13-15) and enzymatic removal of carbohydrate (4) were performed as described pre- viously. Hexose was determined by the anthrone method (16) employing a galactose and mannose standard at a molar ratio of 1:2. Fucose was deter- mined by the reaction with cysteine (17) and sialic acid was determined by reaction with thiobarhituric acid (18). Hexosamines were determined with an amino acid analyzer employing a 20-cm column of PA 35 (Beckman) resin jacketed at 55°C (19). Sam- ples for hexosamine analyses were hydrolyzed with 4 N HCl at 105’C for 6 h and dried ilz vacua over sodium hydroxide. Galactosamine was not detected 0003-9861/83/0205’72-04$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. 572